Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to extracellular stimuli, among many others, and is deregulated in many diseases. Protein phosphorylation is coordinated by the opposing activities of protein kinases and protein phosphatases. In eukaryotic cells, most serine/threonine phosphorylation sites are dephosphorylated by members of the Phosphoprotein Phosphatase (PPP) family. However, we only know for a few phosphorylation sites which specific PPP dephosphorylates them. Although natural compounds such as calyculin A and okadaic acid inhibit PPPs at low nanomolar concentrations, no selective chemical PPP inhibitors exist. Here, we demonstrate the utility of endogenous tagging of genomic loci with an auxin-inducible degron (AID) as a strategy to investigate specific PPP signaling. Using Protein Phosphatase 6 (PP6) as an example, we demonstrate how rapidly inducible protein degradation can be employed to identify dephosphorylation SITES and elucidate PP6 biology. Using genome editing, we introduce AID-tags into each allele of the PP6 catalytic subunit (PP6c) in DLD-1 cells expressing the auxin receptor Tir1. Upon rapid auxin-induced degradation of PP6c, we perform quantitative mass spectrometry-based proteomics and phosphoproteomics to identify PP6 substrates in mitosis. PP6 is an essential enzyme with conserved roles in mitosis and growth signaling. Consistently, we identify candidate PP6c-dependent phosphorylation sites on proteins implicated in coordinating the mitotic cell cycle, cytoskeleton, gene expression, and mitogen-activated protein kinase (MAPK) and Hippo signaling. Finally, we demonstrate that PP6c opposes the activation of large tumor suppressor 1 (LATS1) by dephosphorylating Threonine 35 (T35) on Mps One Binder (MOB1), thereby blocking the interaction of MOB1 and LATS1. Our analyses highlight the utility of combining genome engineering, inducible degradation, and multiplexed phosphoproteomics to investigate signaling by individual PPPs on a global level, which is currently limited by the lack of tools for specific interrogation.

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans , the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C . albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3 Δ/Δ and efg1 Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3 Δ/Δ medium alkalinization phenotype, which is dependent on STP2 , a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1 . Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.

Relapsed/refractory DLBCL remains an incurable disease, and single-agent therapies typically show low response rates and/or transient clinical responses. Oncogenic MYD88 mutations occur in ~25% of DLBCL and drive constitutive NFkB activation, promoting proliferation and survival. Despite its role in tumor biology, targeting MYD88MT by inhibiting or degrading IRAK4 alone, a key component of the MYD88 complex, does not drive significant antitumor activity in preclinical models. One potential reason is the frequent redundant NFkB pathway activation by co-mutations, highlighting the need for combination therapies to effectively target the NFkB pathway in DLBCL. To address this challenge, we have developed IRAKIMiDs, heterobifunctional degraders that simultaneously degrade both IRAK4 and IMiD substrates, as a rational therapeutic combination in a single molecule. IRAKIMiDs show increased antitumor activity in vitro and in vivo in MYD88MT cells as compared to IMiD or IRAK4-targeting alone, highlighting suggesting their potential as single agents in R/R DLBCL.Our lead IRAKIMiD, KT-413, is a potent degrader of IRAK4 (DC50 6nM) and IMiD substrates (Ikaros/Aiolos DC50 2nM), inducing rapid and potent cell killing in vitro and complete and sustained tumor regressions in vivo in MYD88MT models of DLBCL. This activity is superior to the IMiD CC-220, which has similar activity against IMiD substrates (Ikaros/Aiolos DC50 1 nM), supporting the synergistic role of IRAK4 degradation in the context of IMiD biology. We show here that the combined activity of these 2 mechanisms drives a synergistic effect on NFkB and IRF4 signaling with greater downstream effect on NFkB and type 1 interferon (IFN) signaling and cell cycle gene expression than either mechanism alone. In THP1 cells engineered with NFkB and IRF4 reporters, KT-413 but not CC-220 inhibits TLR-stimulated NFkB and IRF4 transcription, supporting a role for IRAK4 but not IMiDs in MYD88-driven survival and proliferation signals. IMiDs have previously been shown to modulate type1 IFN signaling through downregulation of IRF4. We propose that simultaneous targeting of both NFkB and type 1 IFN signaling with KT-413 drives synergistic cell killing in MYD88MT cells. In MYD88MT OCI-Ly10 cells, KT-413 leads to greater IRF4 downregulation, increased type 1 IFN signaling, and preferential downregulation of NFkB pathway and cell cycle transcripts when compared to CC-220. These data support the hypothesis that the synergistic activity of targeting IRAK4 and IMiD substrates by KT-413 in MYD88MT DLBCL is a result of dual targeting of NFkB and IRF4/Type1 IFN through degradation of both IRAK4 and IMiD substrates,driving significantly greater cell killing as compared to either mechanism alone, supporting the potential for the first single agent targeted therapy in MYD88MT DLBCL. KT-413 is on track for initiation of a Phase 1 trial in B cell lymphoma in 2H 2021. Citation Format: Christine R. Klaus, Scott F. Rusin, Kirti Sharma, Samyabrata Bhaduri, Matthew M. Weiss, Alice A. McDonald, Michele F. Mayo, Duncan Walker, Rahul Karnik. Mechanisms underlying synergistic activity in MYD88MTDLBCL of KT-413, a targeted degrader of IRAK4 and IMiD substrate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB118.

An important goal of cancer therapy is to improve patient outcomes by driving to deep and durable tumor responses. The activity of single-agent targeted therapies, such as BTK inhibitors or IMiDs alone, has been modest in relapsed and refractory DLBCL, necessitating the use of combination therapy. Targeted protein degraders are heterobifunctional small molecules that co-opt the endogenous ubiquitin-proteasome system to drive the selective degradation of target proteins. Degraders of IRAK4, a key component of the myddosome complex, show potent and selective IRAK4 degradation and preferential activity in MYD88-mutant (MYD88MT) DLBCL models. Notably, the activity of IRAK4 degradation is superior to IRAK4 kinase inhibition, supporting the essential scaffolding role of IRAK4 in myddosome signaling. We have previously described IRAKIMiDs, novel IRAK4 degraders that utilize an IMiD as a pharmacologically active cereblon binder. These degraders simultaneously degrade both IRAK4 and IMiD substrates and show synergistic antitumor activity over either IRAK4 degraders or IMiDs alone, enabling a therapeutically relevant biological combination within a single small molecule. Here we describe KTX-120, a novel IRAKIMiD development candidate. KTX-120 is an equipotent degrader of both IRAK4 and the IMiD substrates Ikaros and Aiolos in lymphoma model systems with low single-digit nM DC50 for degradation of all substrates. The cell activity of KTX-120 has shown a high dependence on MYD88MT status: across a panel of MYD88MT cell lines, KTX-120 showed consistent and potent cell activity, with IC50 ranging from 7-29nM, whereas in MYD88WT lines, cell activity was poor ranging from 1800-3400nM. In the OCI-Ly10 ABC DLBCL cell line that harbors a MYD88L265P mutation, the cell activity of KTX-120 was associated with degradation of both IRAK4 and Ikaros, supporting the combined IRAK4 and IMiD targeting of this molecule as contributing to cell activity. Notably, the onset of cell death in OCI-Ly10 cells with KTX-120 treatment was rapid, with cells becoming committed to cell death within 72h of exposure, suggesting that continuous exposure to KTX-120 may not be necessary for antitumor activity. We explored the pharmacological activity of KTX-120 in several in vivo model systems. KTX-120 is orally bioavailable and shows dose-proportional exposure in several species. A single oral dose of KTX-120 (10 mg/Kg or 30 mg/Kg) showed significant degradation of both IRAK4 and Ikaros in a dose and time-dependent manner, with degradation of both substrates being sustained for >96h, further supporting the potential for intermittent dosing. To assess this, we have explored the antitumor efficacy of KTX-120 in intermittent dosing schedules. KTX-120 was well tolerated and showed potent antitumor activity in several CDX models of MYD88MT DLBCL, including OCI-Ly10, TMD8 and SUDHL2, achieving regressions in all models in as little as once every 2 weeks. As an example, in OCI-Ly10, a 30mpk dose Q2W drove >80% degradation of both IRAK4 and Ikaros and showed regressions (including CR) by D28. Similar activity and tolerability were seen with both PO and IV dosing at doses that achieve active exposure, enabling the potential for both oral and parenteral dosing. We have further explored the activity of KTX-120 in a collection of DLBCL patient derived xenograft models. KTX-120 shows robust activity (>85% TGI) in 4/5 models of MYD88MT DLBCL and shows no or modest activity in 2/2 models of MYD88WT DLBCL, supporting the preferential activity of this mechanism in patient samples harboring MYD88 activating mutations. Importantly, activity was observed in models with a variety of co-mutations that activate the NFkB pathway, including alterations in, CD79B and TNFAIP3, suggesting that KTX-120 has the potential for activity in MYD88MT lymphoma regardless of other mutations. Collectively, these data support the combined IRAK4 degradation and IMiD activity of KTX-120 has the potential to achieve robust and durable regressions in MYD88MT lymphomas with the increased convenience of an intermittently administered single agent and decreased potential for drug combination challenges. Disclosures Walker: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Klaus:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chen:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bhaduri:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rusin:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Weiss:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Mitochondria are often dubbed “the powerhouse of the cell,” however this gross over‐simplification has arguably led to a paucity of knowledge concerning the many other functions of these incredibly diverse organelles. Fatty acid synthesis (FAS) is an essential cellular process, but it is a little‐known fact even among mitochondrial experts that eukaryotes actually harbor two distinct FAS systems – one in the cytoplasm, and one in the mitochondria. Although mitochondrial FAS (mtFAS) was discovered decades ago, its function(s) in mammals are still unclear. One well known product of the pathway is lipoic acid, an important lipid co‐factor for several mitochondrial dehydrogenases, however we now appreciate that mtFAS plays a far wider role in mitochondrial physiology. Here we show that while mtFAS does not directly contribute to the synthesis of structural lipids like PS, it instead is required for the production of lipids that facilitate assembly and stability of the mitochondrial electron transport chain (ETC). This novel regulatory role of mtFAS occurs via physical interaction of the acyl carrier protein and a family of late‐stage ETC assembly factors known as the LYR proteins. Notably, this effect is separate from lipoic acid production, as Lipt1 knockouts do not display the same strong ETC depletion phenotype. Proteomics and metabolomics data provide intriguing insights into the consequences of mtFAS depletion, and highlight promising future directions towards further understanding the role of this pathway in cellular metabolism.Support or Funding InformationThis work was supported partially by grants from the NIH (GM115174 and GM115129 to JR and GM97645 to SPG) and the Nora Eccles Treadwell Foundation (to JR), as well as HHMI (JR). Support for SMN was also provided by UMDF and ACS postdoctoral fellowships.

Abstract Targeted protein degraders (TPD) are heterobifunctional small molecules that employ the ubiquitin degradation system to drive the selective degradation of target proteins. In approximately one third of B-cell lymphomas, gain of function mutations in MYD88 constitutively activate the myddosome and drive proliferation and survival through NFkB and AP1 signaling. IRAK4, is a key component of the myddosome and IRAK4 TPD show potent and selective IRAK4 degradation and preferential activity in MYD88 mutated B cell lymphoma models. Notably, the activity of IRAK4 degraders is superior to kinase inhibition, supporting the essential scaffolding role of IRAK4 in Myddosome signaling. An important goal of cancer therapy is to drive to deep and durable responses in patients, and this is frequently achieved by combination therapy. It has been suggested that blocking NFkB while simultaneously inducing a Type 1 IFN response with an IMiD (e.g. Lenalidomide Yang et al. (2012) Cancer Cell 21, 6, p723) may drive additive or synergistic antitumor activity. We have developed novel degraders that utilize an IMiD as a cereblon binder. These compounds simultaneously degrade both IRAK4 and IMiD substrates, enabling a therapeutically-relevant biological combination within a single small molecule. Here we show that dual IRAK4 and IMiD based degraders have potent antiproliferative activity across MYD88 mutant, but not MYD88WT lymphoma cell lines, with increased and broader antiproliferative and apoptosis activity relative to selective IRAK4 degraders or IMiDs alone. Several lines of evidence suggest that both IRAK4 and IMiD activity contribute to the overall superior activity of the dual degraders. Proteomic analysis demonstrates degradation of both both IRAK4 and key IMiD substrates, including Ikaros and Ailolos. This activity results in an enhanced Type 1 IFN response signature compared to selective IRAK4 degraders or IMiD alone, suggesting cooperative biological activity. Degradation of both IRAK4 and IMiD substrates corelates with cell activity, consistent with a requirement for both activities to drive cell death. In xenograft models of MYD88 mutant DLBCL, daily oral dosing of lead dual - degraders demonstrates strong antitumor activity in xenograft models of MYD88 mutant lymphoma, including durable complete regressions that can be maintained for several weeks following cessation of dosing. Regressions are associated with degradation of both IRAK4 and IMiD substrates, consistent with the dual-targeting activity of these molecules. Collectively, these data support a strategy to combine IRAK4 degradation and IMiD activity in a single molecule that will have the potential benefits of enhanced therapeutic activity, increased convenience and decreased potential for drug combination challenges, such as DDI. Lead degraders based on this synergistic profile are being advanced towards IND in 2020. Citation Format: Duncan H. Walker, Michele Mayo, Christine Klaus, Haojing Rong, Scott Rusin, Kirti Sharma, Alice McDonald, Veronica Campbell, Joseph Kelleher, Jared Gollob, Nello Mainolfi, Matt Weiss. Degraders targeting both IRAK4 and IMiD substrates show combinatorial effects leading to broader activity with durable and complete regressions in MYD88 mutant lymphoma xenografts in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5222.

STAT3 (signal transducers and activators of transcription 3) is a transcription factor and a member of the STAT protein family that is activated through a variety of different cytokine and growth factor receptors via JAKs, as well as through oncogenic fusion proteins and gain-of-function (GoF) mutations in STAT3 itself. STAT3 hyperactivation and GoF mutations are found in numerous cancers, including clinically aggressive hematologic malignancies with high unmet medical need, such as peripheral T cell lymphomas (PTCLs) (Andersson et al., 2020). We have previously shown that a potent and selective STAT3 heterobifunctional degrader, KTX-201, strongly represses cell growth in models of STAT3-dependent heme malignancies (Csibi et al., 2019). Herein, we report on the cellular mechanisms underlying the anti-tumor effect of STAT3 degradation in PTCL and provide a model for the relationship between pharmacokinetics/ pharmacodynamics (PK/PD) and activity of KTX-201 in vivo. The relationship between STAT3 degradation by KTX-201, anti-tumor mechanism of action and in vivo activity were investigated in anaplastic large T cell lymphoma (ALCL) models, a subset of PTCLs. In vitro, a decrease of STAT3 by 90% for 48hr was required for ALCL cells to commit to death. To identify anti-tumor mechanism(s) of KTX-201 at the systems level, we performed a time-resolved analysis of the proteomic changes of SU-DHL-1 cells undergoing growth inhibition mediated by KTX-201 at GI95. We measured the abundance of 10,000 proteins and confirmed selective degradation of STAT3 by KTX-201 after 8h of treatment. Significant changes in several marker proteins known to be involved in STAT3-mediated proximal signaling in ALCL including SOCS3, Myc and Granzyme B were observed after 16h. Functional annotation analysis of proteins identified pathways that were significantly enriched in at least one time point. Using unsupervised hierarchical clustering of annotations, we found that proteins that increased in abundance over 48h of exposure to KTX-201 were associated with markers of apoptosis and those that decreased in abundance by 24h and 48h were associated with cytokine signaling and cell cycle, respectively. Based on these data, this study identifies inhibition of cytokine signaling, G1 cell cycle arrest and induction of apoptosis as key anti-tumor mechanisms associated with KTX-201 consistent with observed cell phenotypes. STAT3 degradation in tumor was characterized in mice bearing SU-DHL-1 tumors following single dose IV administration. The STAT3 PD response in tumor was correlated with exposures in tumor. At the dose of 25 mg/kg weekly where complete tumor regression was achieved, KTX-201 achieves >90% STAT3 degradation at 24h post dosing in SUDHL1 xenografts. STAT3 degradation was maintained at 90% at 4 days post dosing. The results from the PK/PD study suggests that STAT3 degradation in tumor of >90% is necessary for anti-tumor efficacy in vivo of KTX-201, but only for a limited duration, such as 4 days out of a weekly dosing cycle. Collectively, our data demonstrate that significant STAT3 degradation for a limited time during dosing interval with KTX-201 in ALCL promotes early changes in key signaling nodes involved with proliferation and cytokine stimulation, followed by profound changes in apoptotic proteins. By integrating mechanistic biology with a deep understanding of PK/PD and efficacy, this study provides a foundation for the clinical development of STAT3 degraders using intermittent dosing regimen for treatment of PTCL and other STAT3-dependent heme malignancies. Disclosures Rong: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Csibi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company, Ended employment in the past 24 months. Yang:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rusin:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Shi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chutake:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhu:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ji:Kymera Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Liu:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Li:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Walker:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Desavi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels.

Abstract Signal transducer and activator of transcription 3 (STAT3) has been implicated in multiple aspects of tumorigenesis. In addition to increasing cancer cell proliferation and survival, constitutively activated STAT3 is proposed to regulate cross-talk between tumor, stroma and immune cells to promote immune-evasion. STAT3 activity in tumors promotes the production of immune-suppressive factors that activate STAT3 in diverse immune-cell subsets. Mechanistically, genetic studies support a direct role of activated STAT3 in regulating myeloid cell differentiation to contribute to an immune-suppressed tumor microenvironment (TME) (Kortylewski et al.; Nat. Med. 2005 and Curr. Opin. Immunol. 2008) Therefore, STAT3 is a highly attractive target for immune-oncology. Here, we demonstrate that the degradation of STAT3 with a potent and selective STAT3 degrader reverses immune suppression in preclinical models. KYM-003 is a heterobifunctional molecule that hijacks the ubiquitin-proteasome system (UPS) for rapid STAT3 degradation. KYM-003 robustly degraded STAT3 in both human peripheral blood mononuclear cells (hPBMC)-derived monocytes and lymphocytes with DC50 < 100 nM. Degradation in hPBMCs was highly selective for STAT3 vs >10,000 other detected proteins (including all other STAT family members) as evaluated by deep tandem mass tag proteomics. IL-6 treatment of hPBMCs upregulates STAT3 phosphorylation, resulting in transcription of genes involved in myeloid cell-mediated immune suppression, such as IL-10 and CD163, a marker of M2 macrophage differentiation. Treatment of cells with KYM-003 at DC90 for 6 hours abrogated the IL-6 induced up-regulation of immune-suppressive gene signatures consistent with a role of STAT3 in mediating an immune suppressive environment by regulating macrophages and other myeloid cells. In several tumor cell lines, degradation of STAT3 by KYM-003 led to strong downregulation of PD-L1. Importantly, in the BioMap StroNSCLC (DiscoverRx), a co-culture system composed of NCI-H1299 lung cancer cells, hPBMCs and primary human fibroblasts that models immune-suppressed TME biology, KYM-003 treatment decreased angiogenic and immune-suppressive cytokines, including VEGF and IL-10, and promoted the pro-inflammatory anti-tumor cytokines IL-2, IFNg and TNFa. Lastly, administration of KYM-003 to mice bearing syngeneic tumors exhibited anti-tumor activity as monotherapy in the CT26 syngeneic model. We have shown that targeting STAT3 for degradation may have a role in restoring an immune-permissive environment in tumors by both rescuing the suppressed immunologic microenvironment and directly downregulating immune checkpoint signals in tumor cells. These data support STAT3 degraders as a promising new therapeutic modality as immune-oncology agents.Citations: Kortylewski et al (2005) Nat. Med, 11(12):1314-21. Kortylewski et al (2008) Curr. Opin. Immunol, 20(2):228-33 Citation Format: Alfredo Csibi, Bin Yang, Yogesh Chutake, Karen Yuan, Michele Mayo, Veronica Campbell, Alice McDonald, Scott Rusin, Kirti Sharma, Hari Kamadurai, Henry Li, Mike Sintchak, Sean Zhu, Sharon Townson, Anthony Slavin, Haojing Rong, Phillip Liu, Chris De Savi, Jared Gollob, Duncan Walker, Nan Ji, Nello Mainolfi. A STAT3 selective targeted protein degrader decreases the immunesuppressive tumor microenvironment and drives antitumor activity in preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-088.

Phosphorylation is a post-translational modification with a vital role in cellular signaling. Isobaric labeling-based strategies, such as tandem mass tags (TMT), can measure the relative phosphorylation states of peptides in a multiplexed format. However, the low stoichiometry of protein phosphorylation constrains the depth of phosphopeptide analysis by mass spectrometry. As such, robust and sensitive workflows are required. Here we evaluate and optimize high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) coupled to Orbitrap Tribrid mass spectrometers for the analysis of TMT-labeled phosphopeptides. We determined that using FAIMS-MS3 with three compensation voltages (CV) in a single method (e.g., CV = 40/-60/-80 V) maximizes phosphopeptide coverage while minimizing inter-CV overlap. Furthermore, consecutive analyses using MSA-CID (multistage activation collision-induced dissociation) and HCD (higher-energy collisional dissociation) fragmentation at the MS2 stage increases the depth of phosphorylation analysis. The methodology and results outlined herein provide a template for tailoring optimized FAIMS-based methods.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cells harbor two systems for fatty acid synthesis, one in the cytoplasm (catalyzed by fatty acid synthase, FASN) and one in the mitochondria (mtFAS). In contrast to FASN, mtFAS is poorly characterized, especially in higher eukaryotes, with the major product(s), metabolic roles, and cellular function(s) being essentially unknown. Here we show that hypomorphic mtFAS mutant mouse skeletal myoblast cell lines display a severe loss of electron transport chain (ETC) complexes and exhibit compensatory metabolic activities including reductive carboxylation. This effect on ETC complexes appears to be independent of protein lipoylation, the best characterized function of mtFAS, as mutants lacking lipoylation have an intact ETC. Finally, mtFAS impairment blocks the differentiation of skeletal myoblasts in vitro. Together, these data suggest that ETC activity in mammals is profoundly controlled by mtFAS function, thereby connecting anabolic fatty acid synthesis with the oxidation of carbon fuels. eLife digest In human, plant and other eukaryotic cells, fats are an important source of energy and also play many other roles including waterproofing, thermal insulation and energy storage. Eukaryotic cells have two systems that make the building blocks of fats (known as fatty acids) and one of these systems, called the mtFAS pathway, operates in small compartments known as mitochondria. This pathway only has one known product, a small fat molecule called lipoic acid, which mitochondria attach to several enzymes to allow them to work properly. The main role of mitochondria is to break down fats and other molecules to release chemical energy that powers many processes in cells. They achieve this using large groups of proteins known as ETC complexes. To build these complexes, families of proteins known as ETC assembly factors carefully coordinate the assembly of many proteins and small molecules into specific structures. However, it remains unclear precisely how this process works. Here, Nowinski et al. used a gene editing technique to mutate the genes encoding three enzymes in the mtFAS pathway in mammalian cells. The experiments found that the mutant cells had fewer ETC complexes and seemed to be less able to break down fats and other molecules than ‘normal’ cells. Furthermore, a family of ETC assembly factors were less stable in the mutant cells. These findings suggest that the mtFAS pathway controls how mitochondria assemble ETC complexes. Further experiments indicated that lipoic acid is not involved in the assembly of ETC complexes and that the mtFAS pathway produces another, as yet unidentified, product that regulates this process, instead. MEPAN syndrome is a rare neurological disorder that leads to progressive loss of control of movement, slurred speech and impaired vision in children. Patients with this syndrome have genetic mutations affecting components of the mtFAS pathway, therefore, a better understanding of how the pathway works may help researchers develop new treatments in the future. More broadly, these findings will have important ramifications for many other situations in which the activity of ETC complexes in mitochondria is modified. Introduction Fatty acids play diverse cellular roles, including providing the hydrophobic tails of membrane phospholipids, energy storage in the form of triglycerides, and as cell signaling molecules. Aside from the import of exogenous fatty acids, mammalian cells use the well-known and well-studied cytoplasmic enzyme fatty acid synthase (FASN) to make palmitate, which is further modified to form the diverse array of cellular fatty acids (Smith, 1994). However, it is much less appreciated that mitochondria also harbor a spatially and genetically distinct fatty acid synthesis pathway (mtFAS) (reviewed in Nowinski et al., 2018). In contrast to FASN, which is a very large protein that contains several domains and encompasses all of the enzymatic activities necessary for FAS condensed in a single polypeptide chain, the mtFAS pathway is comprised of at least six enzymes all encoded by separate genes. These enzymes catalyze sequential steps to achieve one cycle of two-carbon addition to a growing acyl chain (Figure 1A). The nascent fatty acids are covalently attached to the mitochondrial acyl carrier protein (ACP), which acts as a soluble scaffold upon which the acyl chains are built. In each cycle of the pathway, malonyl-CoA is converted to malonyl-ACP, which then undergoes a condensation reaction with the growing fatty acyl chain on ACP, extending the chain by two carbons and releasing a CO2 molecule. Thereafter, the remaining enzymes in the pathway must carry out a series of reduction and dehydration reactions to fully reduce the acyl chain to a saturated fatty acid, which is the substrate for further cycles of two-carbon addition. Among the several differences between mtFAS and cytoplasmic FAS, FASN exclusively produces palmitate, whereas mtFAS appears to have at least two major products. Figure 1 with 1 supplement see all Download asset Open asset MtFAS is an essential pathway in mammalian skeletal myoblasts but does not contribute to synthesis of major cellular lipids. (A) Schematic of the mitochondrial fatty acid synthesis pathway and downstream lipoic acid synthesis. (B) Crude isolated mitochondrial fractions from duplicate single cell clones of Mcat, Oxsm, and Mecr mutants, compared with GFP control clonal cell lines, were separated via SDS-PAGE and immunoblotted for the indicated targets. *=Lipoic acid band (reprobe of earlier blot). #=non specific bands C. GFP control and Oxsm mutant cells were infected with retroviral control plasmid (pCtrl) or a plasmid expressing Oxsm off the CMV promoter (pOxsm), plated at equal densities in normal growth medium with either 4.5 g/L glucose or 10 mM galactose and grown for 3 or 4 days, respectively, then stained with crystal violet. (D) Whole cell lysates from stable cell lines generated by infecting Oxsm mutant cells (OxsmΔ) or GFP controls with shRNA constructs targeting FASN (shFASN) or scramble control (shScramble) were separated by SDS-PAGE and immunoblotted for FASN, lipoylated proteins, or tubulin. (E-F) Stable cell lines created in (D) were incubated with U13C-glucose for the indicated number of doublings, harvested, lipids extracted, and analyzed via LC-MS. Shown are quantitation of m+2 isotopologues for two representative phospholipid species, PC 16:0_16:0 and PC 16:0_18:0. †=p < 0.001, ‡=p < 0.0001, error bars are SEM. Although the human genes responsible for each step in the mtFAS pathway have been identified, and their ability to complement the orthologous mutants in yeast has been demonstrated (Autio et al., 2008; Chen et al., 2009; Joshi et al., 2003; Miinalainen et al., 2003; Zhang et al., 2005; Zhang et al., 2003), few loss-of-function studies have examined the consequences of mtFAS deficiency in mammalian systems. Inducible knockout of the mitochondrial malonyl CoA-acyl carrier protein transacylase (Mcat) using a Cre driver that expresses in most tissues in mice results in a severe phenotype characterized by weight loss, reduced muscle strength, and shortened lifespan despite the persistence of residual MCAT protein (Smith et al., 2012). Knockout of the mitochondrial 2-enoyl thioester reductase, Mecr, is lethal in mice due to a placental defect (Nair et al., 2017), while the inducible knockout of Mecr specifically in Purkinje cells leads to loss of this cell population and recapitulates many phenotypes of MePaN syndrome, the human disease caused by Mecr mutation (Gorukmez et al., 2019; Heimer et al., 2016; Nair et al., 2018). Similarly, Mecr knockdown has been shown to inhibit the growth of hepatocellular carcinoma cells (Cai et al., 2019). While these detrimental phenotypes are clear, the molecular consequences of mtFAS loss remain poorly understood. MtFAS currently has one known product: an eight-carbon saturated fatty acid, octanoate, that is subsequently converted to lipoic acid. This important cofactor is required for the catalytic activity of a number of mitochondrial enzymes, most notably pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (Brody et al., 1997; Wada et al., 1997), but also branched chain amino acid dehydrogenase, the H protein of the glycine cleavage system, and 2-oxoadipate dehydrogenase (reviewed in Solmonson and DeBerardinis, 2018). This observation, made more than 20 years ago, has guided mtFAS-focused research for the past two decades. Studies have shown that loss of lipoic acid synthesis and/or failure to efficiently transfer lipoic acid to its target proteins is lethal in mice (Ni et al., 2019; Yi and Maeda, 2005), and attributed other mitochondrial changes as downstream of lipoic acid synthesis (Smith et al., 2012). However, though the mouse studies described above have demonstrated striking phenotypes that result from loss of mtFAS, they fail to distinguish between direct effects of loss of protein lipoylation versus loss of other mtFAS function(s). Although lipoic acid unmistakably has important central functions in mitochondrial metabolism, there is reason to be skeptical that the sole function of mtFAS is the production of lipoic acid. In addition to the eight-carbon precursor for lipoic acid, it is clear that mtFAS also produces longer acyl chains of at least 14 carbons, yet the identity of these lipids and their cellular functions are uncertain (Angerer et al., 2017; Witkowski et al., 2007). Perhaps more surprisingly, these longer acyl chains appear to partially be maintained in attachment to ACP (Angerer et al., 2017), further complicating the question of their cellular functions. We set out to systematically examine the cellular functions of the mtFAS pathway by mutating genes that encode three distinct steps in the pathway, Mcat, Oxsm, and Mecr. For comparison, we also engineered cells with loss of Lipt1, the terminal enzyme in the production of lipoylated enzymes downstream of mtFAS. We found that several phenotypes resulting from loss of the mtFAS pathway are not related to lipoic acid, but must instead be due to loss of other products or functions of mtFAS. In particular, we find that loss of mtFAS, but not lipoic acid synthesis, leads to a profound impairment in the assembly of the mitochondrial electron transport chain machinery, with the attendant consequences on metabolism and cell behaviors. Our data therefore suggest that the mtFAS pathway acts as a key regulator of mitochondrial respiratory metabolism. Results Mitochondrial fatty acid synthesis is an essential pathway that does not contribute to synthesis of cellular lipids The enzymes of mtFAS are ubiquitously expressed in mammalian tissues, with the highest expression in skeletal muscle and heart (Triepels et al., 1999). Therefore, we chose cultured skeletal myoblasts as a model system in which to examine the role of this pathway. We employed a CRISPR/Cas9-based strategy to mutate three genes encoding enzymes in the mtFAS pathway, using two different guide RNA sequences per gene. We targeted Mcat, encoding the malonyl-CoA ACP transacylase, Oxsm, encoding the beta-ketoacyl synthase that condenses malonyl-ACP with the growing acyl chain, and Mecr, encoding the terminal reductase in each cycle of two-carbon unit addition (Figure 1A). It is important to note that because mtFAS is a cycle that requires all enzymes for each stepwise two-carbon addition, loss of each individual enzyme blocks fatty acid synthesis at an early, albeit distinct step. Mcat mutants should be unable to attach any carbon to ACP, whereas Oxsm mutants should be able to produce malonyl-ACP, and Mecr mutants could at most build short four-carbon acyl chains. We observed roughly 70% editing efficiency for all six of the guides utilized (based on T7E1 assays, Figure 1—figure supplement 1A). We found that transfection with Cas9 and sgRNAs targeting mtFAS genes led to smaller colonies at one-week post-single cell sorting relative to control guides (Figure 1—figure supplement 1B). Many of the smallest clones stopped growing and/or did not survive expansion. After screening more than 100 of the surviving single cell clones, we failed to identify a single null mutant for any of the three genes targeted. This finding was not altogether unexpected, given that at least one other knockdown study concluded that NDUFAB1, the mammalian mitochondrial ACP, is essential in HEK293T cells (Feng et al., 2009). Indeed, The Broad Institute’s Depmap lists NDUFAB1 as a ‘common essential’ gene, and the mtFAS genes all display negative gene effects, indicating they are essential in at least some cell lines (Broad, 2020). Taken together, our results strongly support the conclusion that the mtFAS pathway is essential in C2C12 skeletal myoblasts. Although no complete null clones were generated, we were able to isolate several clonal cell lines with markedly decreased abundance of MCAT, OXSM, and MECR. These clones also had no detectable lipoylation of PDH and OGDH subunits (DLAT and DLST, respectively) in isolated mitochondrial fractions (Figure 1B), and grew slowly relative to control clones (Figure 1—figure supplement 1C). Growth of mtFAS mutant cells in both glucose and galactose, the latter of which requires mitochondrial respiration, was normalized by re-expression of the cognate mtFAS gene (Figure 1C, Figure 1—figure supplement 1D and E). MECR has been reported to have dual localization to the cytoplasm in addition to mitochondria (Kim et al., 2014), so we performed sub-cellular fractionation to assess these two MECR populations in our mutant cells. We observed an overall decrease in MECR expression that was most pronounced in the mitochondrial compartment (Figure 1—figure supplement 1F). We thus moved forward with the characterization of these presumably hypomorphic cell lines. As mentioned previously, the canonical function of mtFAS is the production of octanoate, the eight-carbon precursor for lipoic acid synthesis. However, some have speculated that mtFAS might also generate fatty acids that contribute to phospholipid synthesis. Seminal studies found 3-hydroxymyristate as the predominant acyl modification on mitochondrial ACP in fungi (Mikolajczyk and Brody, 1990; Schneider et al., 1997). A more recent effort to identify the predominant lipid species found a variety of medium and long chain acyl species, raising the possibility of multiple products in addition to octanoate (Angerer et al., 2017). Although mtFAS mutant yeast display changes in steady-state lipids, the interpretation of this observation is confounded by other phenotypes of these cells (Schneider et al., 1995). In the only study that examines the role of mtFAS in lipid synthesis in mammals, transient knockdown of ACP did not change the abundance of mitochondrial lipids; however, this does not exclude the possibility that cytoplasmic FAS can compensate for loss of mtFAS (Clay et al., 2016). We therefore set out to test whether mtFAS contributes to cellular phospholipid pools in the presence and absence of the cytosolic fatty acid synthase, FASN. In wild type and Oxsm mutant cell lines, we stably expressed hairpins targeting FASN, which encodes the cytoplasmic fatty acid synthase, or a scrambled control (Figure 1D). We observed no effect of FASN knockdown on protein lipoylation (Figure 1D). We then fed the cells uniformly labeled 13C-glucose and observed labeling in cellular lipids to define the relative contributions of cytoplasmic FAS and mtFAS to various cellular lipid pools. By monitoring accumulation of the m+two isotopologue over time, we observed a dependence on FASN expression, but not mtFAS for the synthesis of new phospholipids (shown are two representative PC species, Figure 1E and F). Higher order isotopologues from additional cycles of elongation by fatty acid synthesis (e.g. m + 4, m + 6, m + 8, and so on) did not show labeling above background in this experiment. Similarly, fatty acid methyl esther analysis (FAMES) showed an effect of FASN knockdown, but not Oxsm mutation, on incorporation of U13C-glucose into c16:0 and c18:0 lipids, which confirmed that the m+2 labeling of phospholipids is likely in the fatty acid tails (Figure 1—figure supplement 1G). These experiments demonstrate that longer acyl chains synthesized by mtFAS do not contribute to cellular fatty acid and phospholipid pools and likely perform some other function. Mutation of mtFAS results in a profound mitochondrial respiratory phenotype and loss of ETC complexes Given the known and hypothesized functions of mtFAS, we next assayed mitochondrial function in the mtFAS mutant cells. We performed a standard Seahorse mitochondrial stress test in high glucose medium and found that the mtFAS mutant cells displayed a roughly 30–40% decrease in basal respiration rate compared with controls (Figure 2A). More strikingly, the mtFAS mutants exhibited a lack of spare respiratory capacity, with FCCP-stimulated uncoupled respiration rates that were similar to the initially measured basal respiration (Figure 2A). Interestingly, Oxsm and Mecr mutants exhibited an increased basal extracellular acidification rate, suggesting that these cell lines compensate for decreased respiration via increased glycolysis, whereas Mcat mutants do not (Figure 2—figure supplement 1A). In agreement with the observed decrease in cellular respiration, mtFAS mutants also show a markedly decreased mitochondrial membrane potential, as measured by the ratio of MitoTracker Red to MitoTracker Green fluorescence (Figure 2B,C and Figure 2—figure supplement 1B). Figure 2 with 1 supplement see all Download asset Open asset MtFAS mutants display profound loss of mitochondrial respiration and ETC complexes. (A) Cells from three clones of each of the indicated genotypes were seeded in eight wells of a 96-well seahorse plate and allowed to adhere overnight, then equilibrated and treated with the indicated drugs following standard mitochondrial stress test protocols from the manufacturer to determine Oxygen Consumption Rate (OCR). #=p < 0.01, †=p < 0.001, ‡=p < 0.0001 all comparisons are to GFP control, error bars are SEM. (B) Cells of the indicated genotype were seeded in chambered coverglass slides, stained with Mitotracker Red and Mitotracker Green, and imaged. (C) Ratio of MitoTracker Red to MitoTracker Green fluorescence of cells from 30 fields of view quantified using Fiji ImageJ. *=p < 0.05, †=p < 0.001, ‡=p < 0.0001 all comparisons are to GFP control, error bars are SD. (D) Mitochondrial lysates generated from the indicated cell lines were normalized for total protein by BCA assay, incubated with 1% digitonin, then separated by blue-native PAGE and immunoblotted with the indicated antibodies. Concurrent decreases in mitochondrial membrane potential and respiration can have several causes ranging from altered substrate utilization and TCA cycle activity to decreased expression and/or activity of electron transport chain (ETC) complexes. Therefore, we examined OXPHOS complex expression and assembly via blue-native PAGE analysis. Strikingly, fully assembled ETC complex I (CI), complex II (CII), and complex IV (CIV) were almost completely absent in mtFAS mutant mitochondria (Figure 2D). Complex V was also decreased in abundance, albeit to a lesser magnitude than that seen for CI, CII, and CIV. In contrast, complex III (CIII) was relatively unaffected by loss of mtFAS, although CIII-containing supercomplexes (SC) were absent, likely resulting from loss of CI and CIV. Furthermore, while CIII abundance and assembly were relatively unaffected, its activity was reduced in each of the mtFAS-deficient cells (Figure 2—figure supplement 1C). Altogether, these results depict a severe respiratory deficiency in mtFAS mutant cells, characterized by the striking loss of stably assembled OXPHOS complexes in mtFAS mutant mitochondria. Notably, this contrasts sharply with FASN, deletion of which does not affect fatty acid oxidation in muscle (Funai et al., 2013). mtFAS supports ETC assembly via the post-translational stabilization of LYRM proteins To further examine the mechanisms underlying loss of ETC complexes in mtFAS mutant cells, we performed an unbiased quantitative proteomics experiment on duplicate whole cell lysates from three mtFAS mutant clones (one Oxsm and two Mecr mutants) and two control clones. Overall, there was no general trend for the steady-state abundance of mitochondrial proteins, with 77 proteins being statistically more abundant and 34 proteins being decreased in abundance (Figure 3A). We also observed that the majority of the proteins that comprise OXPHOS complexes were likewise similar in abundance between control and mtFAS mutant cells, including those in Complexes I, II and IV (Figure 3A,B), despite the decrease in abundance of the completely assembled complexes by blue-native PAGE (Figure 2D). However, a subset of eight OXPHOS proteins displayed significantly decreased abundance in both Oxsm and Mecr mutant cell lines compared to controls, including NDUFA6, NDUFA12, NDUFS4, NDUFS6, SDHB, COX5a, COX5b, and ATPIF1 (Figure 3A,B). To test whether these changes were transcriptional, we performed RNAseq analysis on control and mtFAS mutant cells. However, we found no significant changes in the abundance of transcripts encoding ETC component proteins (Figure 3C and Figure 3—figure supplement 1A–C), implying that the observed decrease in abundance of these proteins is likely due to post-translational regulation. Figure 3 with 1 supplement see all Download asset Open asset Posttranslational loss of ETC components in mtFAS mutants is specific to LYR proteins and their targets. (A-B) Duplicate samples from the indicated cell lines were grown under proliferative conditions and subjected to TMT labeling and quantitative proteomics analysis. (A) Volcano plot of compiled Mecr clones vs. GFP controls showing all proteins (gray), mitochondrial proteins (blue), and electron transport chain subunits (ETC, red). Dashed gray lines indicate cutoffs for significance at -log10(p-value) = 1.3 and log2(Fold Change) = +/- 0.59. (B) Heatmap depicting log2(Fold Change) of OXPHOS subunits in the indicated cell lines. (C) Quadruplicate samples from mtFAS mutant cells and controls were grown under proliferative conditions. Total RNA was isolated, used as input for mRNA library prep, and sequenced. Resulting data were aligned to the mouse genome and analyzed for differential expression. ETC subunit-encoding transcripts are shown in red vs. all other transcripts (gray). (D-G) Relative abundance of the indicated LYR proteins or their targets in the indicated cell lines from the quantitative proteomics experiment described in (A-B) #=p < 0.01, †=p < 0.001, ‡=p < 0.0001, error bars are SD. All statistical comparisons shown are between mtFAS mutants and GFP-1 clone; p-values when compared with GFP-2 clone were similar or smaller than when compared with GFP-1 clone. (H) Crude mitochondrial lysates generated from the indicated cell lines by differential centrifugation were normalized for total protein by BCA assay, separated by SDS-PAGE, and immunoblotted with the indicated antibodies. The two proteins that were most decreased in abundance, NDUFA6 and SDHB (Figure 3D,E), also stood out because of their relationships with the leucine-tyrosine-arginine motif (LYRM) protein family, a family of small proteins chiefly comprised of late-stage ETC assembly factors (Angerer, 2015). In many cases, such as for Complex II, LYRM proteins (SDHAF1 and SDHAF3) bind to and facilitate the insertion of a target protein (in this case SDHB) in the final stage of ETC complex assembly (Ghezzi et al., 2009; Na et al., 2014). In other cases, such as for Complex I, the LYRM proteins NDUFA6 and NDUFB9 are actually stable subunits of the fully assembled complex (Fiedorczuk et al., 2016; Zhu et al., 2016). LYRM proteins are also found as a stable component of the NFS-containing iron-sulfur cluster biogenesis (ISC) complex (LYRM4) as well as in an assembly intermediate of the mitochondrial ribosome (AltMid51) (Boniecki et al., 2017; Brown et al., 2017). High-throughput interactomics studies in mammalian systems and targeted experiments in yeast have identified physical interactions between ACP and several LYRM proteins (Floyd et al., 2016; Huttlin et al., 2015; Majmudar et al., 2019; Van Vranken et al., 2018). Interestingly, the ACP protein found in complex with LYRM proteins appears often to have maintained its acyl modification, which is generated through the sequential actions of the mtFAS enzymes as described above (Angerer et al., 2014; Cory et al., 2017; Runswick et al., 1991; Van Vranken et al., 2016). Studies have shown that the acyl chain on ACP is intimately involved in these physical interactions, folding into the middle of the LYRM proteins, but the role of ACP acylation in its varied functions is mostly untested (Angerer et al., 2017; Boniecki et al., 2017; Cory et al., 2017; Fiedorczuk et al., 2016; Zhu et al., 2016). In addition to the structurally verified ACP-LYRM interactions, three other LYRM family members with known target proteins have been suggested to interact with ACP based on high-throughput interactomics studies: SDHAF1 and SDHAF3, discussed above, and LYRM7, which mediates the addition of UQCRFS1 in the last step of complex III assembly (Floyd et al., 2016; Huttlin et al., 2015; Na et al., 2014; Sánchez et al., 2013). Thus, we hypothesized that the decreased abundance of SDHB (Figure 3E) could be explained by interaction of ACP with SDHAF1 and/or SDHAF3. To verify whether ACP in fact interacts with these additional LYRM family members, we expressed epitope-tagged variants of ACP and each LYRM in cells and confirmed their physical interaction via co-immunoprecipitation and immunoblotting (Figure 3—figure supplement 1D). Along with other published data (Van Vranken et al., 2018), the finding that NDUFA6, a known LYRM protein, and SDHB, the target of two known LYRM proteins, exhibit decreased abundance in mtFAS mutant cell lines supports the hypothesis that interaction with an acylated ACP is required for the complex assembly functions of these LYRM proteins. This finding prompted us to examine the abundance of other LYRM proteins and their targets in the mtFAS mutant cell lines. Indeed, LYRM4 and its target NFS1 were also significantly decreased in abundance in our whole cell proteomics dataset, which was confirmed by western blot, implying that LYRM4 also requires acylated ACP for the stability and function of itself and NFS1 (Figure 3F–H). Interestingly, NDUFB9, the other CI LYRM, and UQCRFS1, a subunit of CIII and the target of LYRM7, were not significantly decreased in abundance in mtFAS mutant cells (Figure 3—figure supplement 1E,F), implying that either the interactions between ACP and these LYRM proteins is not dependent on acylation and mtFAS, or that other compensatory mechanisms are at play. We performed immunoblot analysis of UQCRFS1 and found that it was also decreased in abundance similarly to other LYR targets, perhaps explaining the observed decrease in CIII activity despite the partial maintenance of complex assembly (Figure 3H). Finally, ACP has also been found to interact with an assembly intermediate of the mitochondrial ribosome through a novel LYRM protein called altMiD51 and its target, MALSU1 (Brown et al., 2017). Brown et al. proposed that this interaction might negatively regulate mitochondrial translation because MALSU1, altMiD51, and ACP bind the large mitochondrial ribosomal subunit in a way that precludes small subunit binding, but the role of ACP acylation in this interaction is unclear (Brown et al., 2017; Dibley et al., 2020; Rathore et al., 2018). Despite this proposed role of ACP in mitochondrial translation, we found that the abundance of mitochondrially encoded proteins was unchanged in the mtFAS mutant cell lines (Figure 3—figure supplement 1G). Importantly, this includes MTCO1 and MTCO2, two subunits of CIV, strongly implying that the diminution of CIV in mutant cells does not result from loss of mitochondrial translation. Interestingly, however, MALSU1 was significantly decreased in abundance (Figure 3—figure supplement 1H), which raises the possibility that MALSU1 downregulation might be a compensatory adaptation that the cells make to avoid collapse of mitochondrial translation upon loss of mtFAS. Impairment of mtFAS induces reductive labeling of TCA cycle intermediates The profound defects observed in mitochondrial respiration led us to examine mitochondrial and cellular metabolism more comprehensively in our mtFAS mutants. We first performed steady-state metabolomics analysis on control and mtFAS mutant cells. Purines were among the most significantly depleted metabolites in mtFAS mutants, but we observed no change in pyrimidines (Figure 4—figure supplement 1A,B, all measured metabolites in Supplementary file 1). Purine depletion may be a result of decreased activity of another lipoylated protein – the glycine cleavage H protein, which plays a role in purine biosynthesis (Fujiwara et al., 1991). mtFAS mutants exhibited robust pyruvate accumulation, in agreement with the decreased lipoylation and reduced activity of PDH, but only a minor and statistically insignificant increase in intracellular lactate (Figure 4A and Figure 4—figure supplement 1C). Similarly, no significant changes were observed in the abundance of glutamine or glutamate (Figure 4—figure supplement 1D,E). In contrast, the TCA cycle intermediates citrate, fumarate, a

Abstract Purpose: Targeted protein degradation is a novel therapeutic modality that holds the promise to target previously “undruggable” proteins by harnessing the innate ubiquitin proteasome system in cells. In this study we describe the discovery and characterization of KYM-003, a novel, selective degrader of the oncogenic transcription factor STAT3 that shows potent antitumor activity in vitro and in vivo in STAT3-dependent models. Introduction: STAT3 is a transcription factor downstream of several signaling events including the IL-6-JAK pathway. Activating mutations and aberrant STAT3 activation are found in numerous cancers and STAT3 activation has been directly linked to the promotion of cancer cell survival, proliferation and immune evasion, making it a highly attractive target for oncology. Potent and selective agents specifically and directly targeting STAT3 have remained elusive, however. Herein we report the discovery of a potent and selective STAT3 degrader, KYM-003, which displays strong anti-tumor activity in models of heme malignancies. Methods: The binary binding of KYM-003 to STAT3 and the E3 ligase were characterized in fluorescence polarization assays. An AlphaLISA immunoassay was used to measure the formation of the E3-KYM-003-STAT3 ternary complex. Ubiquitination of endogenous STAT3 was evaluated in human cell lysates in the presence of KYM-003. STAT3 levels in multiple primary cells, cell lines, and xenograft tumor tissues were quantified by immunoassays. STAT3 signaling was monitored through its target gene levels by RT-qPCR. Cell viability was monitored by CellTiter-Glo. Tumor xenograft studies were conducted by implanting human cancer cell lines into immune-compromised mice and assessment of tumor volume changes. Key data: KYM-003 degrades STAT3 via an E3 ligase-dependent mechanism. KYM-003 potently binds to and demonstrated strong cooperativity in promoting the formation of the STAT3-KYM-003-E3 ligase ternary complex. In a biochemical assay with A549 cell lysates, KYM-003 potently induced ubiquitination of endogenous STAT3. KYM-003 robustly degraded STAT3 in numerous primary cells or cell lines with DC50 < 100 nM. Degradation was highly selective for STAT3 vs >10,000 other detected proteins (including all other STAT family members) in A549 and hPBMCs. Degradation of STAT3 by KYM-003 led to strong downregulation of STAT3 targets gene expression, such as SOCS3, MYC, and PIM1 Importantly, total STAT3 and pSTAT3 levels in tumors were reduced by >90% for at least 24 hours after a single dose of KYM-003 and repeated dosing of KYM-003 showed dose-dependent antitumor activity in xenograft models of heme maliganancies. Conclusion: KYM-003 is a potent and selective STAT3 degrader that demonstrated strong anti-tumor activity in heme cancer models. These data support STAT3 degraders as a promising new therapeutic opportunity. Citation Format: Fred Csibi, Nan Ji, Bin Yang, Karen Yuan, Michele Mayo, Haojing Rong, Scott Rusin, Kirti Sharma, Christine Loh, Henry Li, Sharon Townson, Hari Kamadurai, Jesse Chen, Duncan Walker, Jared Gollob, Nello Mainolfi. Discovery of KYM-003, a potent and selective STAT3 degrader with antitumor activity in heme malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C054. doi:10.1158/1535-7163.TARG-19-C054

The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP.dkfz.de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.

Purpose: This work assessed the antitumor activity of selective small molecule IRAK4 degraders in human ABC DLBCL cell lines in vitro and in tumor xenograft models in vivo, alone and in combination with BTK inhibition. Introduction: ABC DLBCL comprises approximately 45% of DLBCL and has a worse outcome with R-CHOP chemotherapy compared to GCB DLBCL. Activating mutations in MYD88 occur in 30-40% of ABC DLBCL; L265P, the most prevalent MYD88 mutation, causes constitutive assembly and activation of the Myddosome. IRAK4 kinase and scaffolding functions are essential for full signaling through the Myddosome to NFκB and MAPK pathways. Kymera Therapeutics is using a chemical knockdown strategy to develop heterobifunctional small molecule IRAK4 degraders, exemplified by KYM-001, for the treatment of MYD88-driven lymphomas. Methods: IRAK4 in human PBMC, ABC DLBCL cell lines and xenografts was quantified by immunoassays or targeted MS/MS. Myddosome signaling was monitored by mRNA and phosphoprotein endpoints. Cell viability and cell cycle were monitored by flow cytometry. Tumor xenograft studies were conducted by implanting human ABC DLBCL lines into immunocompromised mouse strains and assessing tumor volume. Key data: KYM-001 led to potent E3 ligase-dependent degradation of IRAK4. Notably, KYM-001 more effectively inhibited TLR-activated Myddosome signaling compared to IRAK4 kinase inhibitors in human PBMC. Degradation was highly selective for IRAK4 vs >10,000 other detected proteins in the MYD88 L265P mutant ABC DLBCL line OCI-LY10. IRAK4 degradation by KYM-001 resulted in cell cycle inhibition and apoptosis within 48-72 h in ABC DLBCL, with preferential activity in MYD88-mutant vs MYD88-WT cell lines. Oral dosing of KYM-001 showed dose-dependent antitumor activity in several mouse xenograft models of human MYD88-mutant ABC DLBCL at tolerated doses and schedules. In the OCI-LY10 model, tumor regression was associated with >80% degradation of IRAK4, establishing the pharmacodynamic effect required for maximal efficacy. Since alterations in BCR signaling and MYD88 frequently co-occur in B-cell malignancies, we investigated the potential for combined activity of IRAK4 degradation and BTK inhibition. In the OCI-LY10 xenograft model, which has activating mutations in both CD79B and MYD88, BTK inhibition with ibrutinib had an additive effect on KYM-001 antitumor activity. Conclusions: KYM-001 is a first-in-class, potent, selective and orally active IRAK4 degrader that causes tumor regression in ABC-DLBCL models. Degradation of IRAK4 removes both the kinase and scaffolding functions of IRAK4, and may be superior to kinase inhibition alone. These data support IRAK4 degraders as a promising new therapeutic opportunity for MYD88-driven lymphoma, both alone and in combination with other targeted approaches such as BTK inhibition. Citation Format: Joseph F. Kelleher, Veronica Campbell, Jesse Chen, Jared Gollob, Nan Ji, Hari Kamadurai, Christine Klaus, Henry Li, Christine Loh, Alice McDonald, Haojing Rong, Scott Rusin, Kirti Sharma, Dominico Vigil, Duncan Walker, Matt Weiss, Karen Yuan, Yi Zhang, Laurent Audoly, Nello Mainolfi. KYM-001, a first-in-class oral IRAK4 protein degrader, induces tumor regression in xenograft models of MYD88-mutant ABC DLBCL alone and in combination with BTK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-272.

Targeted protein degradation mediated by small molecule degraders represents a new and exciting therapeutic modality to target difficult-to-drug oncogenic proteins including transcription factors. These molecules bind to both the target protein and an E3 ligase, enabling the formation of a ternary complex that leads to ubiquitination and subsequent degradation of the target protein by the proteasome. STAT3 (signal transducers and activators of transcription 3) is a transcription factor and a member of the STAT protein family. In response to cytokines and growth factors, STAT3 is phosphorylated by receptor-associated serine/threonine kinases, and phosphylated STAT3 (pSTAT3) then forms dimers that translocate into the nucleus, binds to DNA, and regulate transcription. STAT3 is frequently mutated and activated in numerous cancers including clinically aggressive hematologic malignancies with high unmet medical need. Mechanistically, aberrant activation of STAT3 has been directly linked to the promotion of cancer cell survival, proliferation and immune evasion, thus making it a highly attractive target for oncology. Potent and selective agents specifically and directly targeting STAT3 have remained elusive, however. Herein we report the discovery of a potent and selective STAT3 heterobifunctional degrader, KYM-003, which displays strong anti-tumor activity in models of STAT3-dependent heme malignancies. KYM-003 degrades STAT3 via an E3 ligase-dependent mechanism. It strongly binds to STAT3 and a E3 ligase, leading to the formation of the a productive ternary complex, which leads to ubiquitination of STAT3 and subsequent proteasomal degradation. KYM-003 robustly degraded STAT3 in a number of primary cells or cell lines with DC50 < 100 nM. Degradation was highly selective for STAT3 vs >10,000 other detected proteins (including all other STAT family members) in cell lines and human PBMCs. Degradation of STAT3 by KYM-003 led to significant downregulation of STAT3 target genes, such as SOCS3, MYC, and PIM1. Importantly, total STAT3 and pSTAT3 levels in tumors were reduced by >90% for at least 24 hours after a single dose of KYM-003 and repeated dosing of KYM-003 showed dose-dependent antitumor activity in xenograft models of heme malignancies. Collectively, our data demonstrates that KYM-003 is a potent and selective STAT3 degrader that exhibited strong anti-tumor activity in vitro and in vivo. These data support STAT3 degraders as a new and exciting therapeutic opportunity in heme malignancies. Disclosures Csibi: kymera Therapeutics: Employment, Equity Ownership. Ji:Kymera Therapeutics: Employment, Equity Ownership. Yang:Kymera Therapeutics: Employment, Equity Ownership. Yuan:Kymera Therapeutics: Employment, Equity Ownership. Mayo:kymera Therapeutics: Employment, Equity Ownership. Rong:Kymera Therapeutics: Employment, Equity Ownership. Rusin:Kymera Therapeutics: Employment, Equity Ownership. Sharma:kymera Therapeutics: Employment, Equity Ownership. Loh:Kymera Therapeutics: Employment, Equity Ownership. Li:Kymera Therapeutics: Employment, Equity Ownership. Townson:Kymera Therapeutics: Employment, Equity Ownership. Chen:kymera therapeutics: Employment, Equity Ownership. Kamadurai:Kymera Therapeutics: Employment, Equity Ownership. Walker:Kymera Therapeutics: Employment, Equity Ownership. Gollob:Kymera Therapeutics: Employment, Equity Ownership. Mainolfi:Kymera Therapeutics: Employment, Equity Ownership.

Connections between the protein kinases that function within complex cell polarity networks are poorly understood. Rod-shaped fission yeast cells grow in a highly polarized manner, and genetic screens have identified many protein kinases, including the CaMKK-like Ssp1 and the MARK/PAR-1 family kinase Kin1, that are required for polarized growth and cell shape, but their functional mechanisms and connections have been unknown [1-5]. We found that Ssp1 promotes cell polarity by phosphorylating the activation loop of Kin1. Kin1 regulates cell polarity and cytokinesis through unknown mechanisms [4-7]. We performed a large-scale phosphoproteomic screen and found that Kin1 phosphorylates itself and Pal1 to promote growth at cell tips, and these proteins are interdependent for localization to growing cell tips. Additional Kin1 substrates for cell polarity and cytokinesis (Tea4, Mod5, Cdc15, and Cyk3) were also phosphorylated by a second kinase, the DYRK family member Pom1 [8]. Kin1 and Pom1 were enriched at opposite ends of growing cells, and they phosphorylated largely non-overlapping sites on shared substrates. Combined inhibition of both Kin1 and Pom1 led to synthetic defects in their shared substrates Cdc15 and Cyk3, confirming a non-redundant functional connection through shared substrates. These findings uncover a new Ssp1-Kin1 signaling pathway, and define its functional and mechanistic connection with Pom1 signaling for cell polarity and cytokinesis. These kinases are conserved in many eukaryotes including humans, suggesting that similar connections and mechanisms might operate in a broad range of cells.

Protein phosphatase 1 (PP1) is a highly conserved protein phosphatase that performs most of the serine- and threonine-dephosphorylation reactions in eukaryotes and opposes the actions of a diverse set of serine and threonine (Ser-Thr) protein kinases. PP1 gains substrate specificity through binding to a large number (>200) of regulatory proteins that control PP1 localization, activity, and interactions with substrates. PP1 recognizes the well-characterized RVxF binding motif that is present in many of these regulatory proteins, thus generating a multitude of distinct PP1 holoenzymes. We showed that a subset of the RVxF binding motifs, in which x is a phosphorylatable amino acid (RV[S/T]F), was phosphorylated specifically during mitosis and that this phosphorylation event abrogated the interaction of PP1 with the regulatory protein. We determined that this phosphorylation was primarily governed by the mitotic protein kinase Aurora B and that high phosphorylation site stoichiometry of these sites maintained the phosphorylation of PP1 substrates during mitosis by disrupting the assembly of PP1 holoenzymes. We generated an antibody that recognizes the phosphorylated form of the RV[S/T]F motif (RVp[S/T]F) and used it to identify known PP1 regulatory proteins (KNL1, CDCA2, and RIF1) and multiple proteins that could potentially act as PP1 binding partners (UBR5, ASPM, SEH1, and ELYS) governed by this mechanism. Together, these data suggest a general regulatory mechanism by which the coordinated activities of Aurora B and PP1 control mitotic progression.