Abstract The antiapoptotic protein, myeloid cell leukemia-1 (Mcl-1), contributes to the pathophysiology of acute myeloid leukemia (AML) and certain B-cell malignancies. Tumor dependence on Mcl-1 is associated with resistance to venetoclax. Voruciclib, an oral cyclin-dependent kinase (CDK) inhibitor targeting CDK9, indirectly decreases Mcl-1 protein expression and synergizes with venetoclax in preclinical models. This dose escalation study evaluated voruciclib in patients with previously treated hematologic malignancies. Initially, voruciclib was administered daily, continuously, on a 28-day cycle (group 1). After 2 patients with prior allogeneic stem cell transplantation had a dose-limiting toxicity (DLT) of interstitial pneumonitis at 100 mg, voruciclib administration was changed to days 1 to 14 of a 28-day cycle (group 2). Forty patients, 21 with AML and 19 with B-cell malignancies, were enrolled. Patients had a median of 3 prior lines of therapy (range, 1-8). Dose escalation in group 2 was stopped at 200 mg, a dose that achieved plasma concentrations sufficient for target inhibition, without DLTs observed. The most common adverse events were diarrhea (30%), nausea (25%), anemia (22%), fatigue (22%), constipation (17%), dizziness (15%), and dyspnea (15%). In AML, 1 patient achieved a morphologic leukemia-free state, and 2 had stable disease. Voruciclib treatment led to a decrease in MCL1 messenger RNA expression, downregulation of myelocytomatosis (MYC) and NF-κB transcriptional gene sets, and reduced phosphorylation of RNA polymerase 2. Voruciclib on intermittent dosing was well tolerated, with no DLTs, paving the way for evaluation of the combination of voruciclib with venetoclax for patients with previously treated AML. This trial was registered at www.clinicaltrials.gov as #NCT03547115.

The antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) promotes cell survival in acute myeloid leukemia (AML), and its overexpression is associated with resistance to venetoclax. Voruciclib, an oral cyclin-dependent kinase 9 inhibitor, indirectly decreases Mcl-1 protein expression and has synergistic activity with venetoclax in AML preclinical models. We hypothesized that voruciclib in combination with venetoclax would induce responses in patients with AML with disease progression after venetoclax therapy. This dose-escalation study evaluated voruciclib administered on days 1 to 14 of 28-day cycles with venetoclax daily. The study enrolled 41 adult patients with AML after failure of previous standard therapies. Patients had a median of 2 (range, 1-7) previous lines of therapy, 19 patients (46%) had ≥3 previous lines of therapy, and 39 (95%) had previous venetoclax. No dose-limiting toxicities were reported in 7 dose levels evaluated. The most common adverse events were nausea (34%), febrile neutropenia (32%), diarrhea (22%), dyspnea (22%), hypokalemia (22%), and thrombocytopenia (22%). Antileukemic activity was observed in 10 (24%) patients, including 3 with complete marrow remission and 7 with stable disease lasting ≥3 months. We observed a rebound of circulating blasts during the 14 days of single-agent venetoclax dosing in 40% of evaluable patients. Mcl-1 protein expression and RNA polymerase II Ser-2 phosphorylation decreased on voruciclib. Overall, the combination of voruciclib with venetoclax was tolerable in patients with relapsed/refractory AML, had antileukemic activity, and showed on-target effects in heavily pretreated patients with disease progression after venetoclax. This trial was registered at www.ClinicalTrials.gov as #NCT03547115.

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 enhanced VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, a purine biosynthesis inhibitor, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired AraC resistance showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. In vivo studies revealed significantly prolonged survival upon combination therapy of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia compared to the vehicle control. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.

Preserving proteostasis is a major survival mechanism for cancer. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is a key oncogenic kinase that directly activates the transcription factor heat-shock factor 1 (HSF1) and the 26S proteasome. Targeting DYRK2 has proven to be a tractable strategy to target cancers sensitive to proteotoxic stress; however, the development of HSF1 inhibitors remains in its infancy. Importantly, multiple other kinases have been shown to redundantly activate HSF1 that promoted ideas to directly target HSF1. The eventual development of direct HSF1 inhibitor KRIBB11 suggests that the transcription factor is indeed a druggable target. The current study establishes that concurrent targeting of HSF1 and DYRK2 can indeed impede cancer by inducing apoptosis faster than individual targetting. Furthermore, targeting the DYRK2-HSF1 axis induces death in proteasome inhibitor-resistant cells and reduces triple-negative breast cancer (TNBC) burden in ectopic and orthotopic xenograft models. Together the data indicate that cotargeting of kinase DYRK2 and its substrate HSF1 could prove to be a beneficial strategy in perturbing neoplastic malignancies.

Phosphatidylinositol-3 kinase (PI3K) delta signaling is essential for CLL B cell survival and proliferation. Several PI3K inhibitors are approved for treatment of CLL, but PI3K inhibitor-induced alterations in T cell function have been correlated with immune-related adverse events (irAEs). Zandelisib is a novel selective PI3Kδ inhibitor in clinical development with an intermittent dosing schedule for treatment of B cell malignancies. Here, we investigated the dynamic immunomodulatory properties of zandelisib alone or in combination with BTK inhibitor ibrutinib in normal human T cells, as well as the impact of continuous dosing of zandelisib on effector and regulatory immune cells, irAEs, and survival in a preclinical CLL murine model. Normal human T cells treated ex vivo with zandelisib, ibrutinib, or both were assessed for phenotype and function. A CLL model was initiated by transferring leukemic EμTCL1 splenocytes into syngeneic wildtype recipients. CLL-bearing mice were treated with vehicle or zandelisib (50 mpk bid or 100 mpk qd) for 3 weeks (n=5 per group). Antitumor efficacy, T cell and myeloid subsets in blood, bone marrow and spleen were analyzed. Survival was also observed in a separate cohort of mice treated with zandelisib (100 mpk qd). T cell proliferation was impaired by zandelisib (250nM) and ibrutinib (100 nM) alone, with the combination further reducing T cell proliferation. Zandelisib decreased expression of suppressive markers PD-1, CTLA-4, GITR and CD39 on iTregs; however, no further decrease was observed when zandelisib was combined with ibrutinib. Zandelisib also reduced normal T cell activation and expression of immune checkpoints PD-1 and CTLA-4 on CD4+ T cells and dose dependently inhibited T cell memory differentiation ex vivo, increasing the naïve/central to effector memory ratio. Combination with ibrutinib did not further affect T cell activation, checkpoint expression or memory differentiation. In the CLL murine model, no meaningful difference was seen between bid or qd administration of zandelisib in the reduction of tumor burden (CD19+ CD5+ CLL cells). Zandelisib treatment reduced activated T cells, antigen-experienced T cells, and Tregs. Increased naïve/central to effector memory ratio was observed in treated mice, and PD-1 was downregulated on memory T cells, indicating less T cell exhaustion. Zandelisib decreased macrophages in the spleen but did not impact myeloid-derived suppressor cells. Zandelisib treatment improved survival of CLL-bearing mice significantly when compared to vehicle group (median survival of zandelisib group 140 days vs. 105 days vehicle). In conclusion, zandelisib treatment reduced Tregs, prevented terminal memory differentiation, and T-cell exhaustion—features that have been shown to permit CLL immune evasion—demonstrated antitumor efficacy, and improved overall survival in a preclinical CLL model. Citation Format: Kamira Maharaj, Melanie Mediavilla-Varela, John J. Powers, Sandra Wiley, Angimar Uriepero, Wael Gamal, Kun Jiang, Eva Sahakian, Javier Pinilla-Ibarz. Efficacy and immune profiling of PI3K delta inhibitor zandelisib (ME-401) in a preclinical chronic lymphocytic leukemia (CLL) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5496.

Abstract Acute myeloid Leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. Despite chromosomal and genetic heterogeneity, AML are uniformly characterized by increased reliance on oxidative phosphorylation (OXPHOS). This key metabolic hallmark of leukemia was recently reported as a feature of resistance to Cytarabine (AraC)-based therapy. Also, an aggressive phenotype and poor response to chemotherapy is associated with increased levels of Bcl-2. Despite the introduction of the Bcl-2 inhibitor venetoclax (VEN), the overall survival, particularly in older patients, remains poor. Thus, approaches to improve the sensitivity of leukemic cells to AraC-based or Bcl-2 based therapies are urgently needed.Here, we investigated the preclinical activity of ME-344, a novel isoflavone OXPHOS inhibitor, on AML cell lines and relapsed/refractory (R/R) patient samples in vitro and examined the efficacy of ME-344 in combination with VEN in Ara-C sensitive and resistant AML cell lines and patient-derived xenografts (PDX) both in vitro and in vivo.ME-344 (0-300 nM, 24 hr) significantly reduced viability of AML cell lines with EC50 of 75-100 nM and R/R AML patient samples with EC50 of 200-300 nM respectively. The cytotoxic response in AML was enhanced when ME-344 was combined with VEN, producing strong synergistic viability reduction and induction of apoptosis, as evidenced by Annexin V assay and an increased level of cleaved caspase 3 and PARP (immunoblotting). The dual inhibition of OXPHOS/Bcl-2 reduced Mcl-1 levels and showed efficacy in Mcl-1 overexpressingand Ara-C resistant AML models.Functional metabolic characterization of AML by transcriptomics and mass spectrometry demonstrated that ME-344 effectively inhibited biosynthetic pathways of nucleotides uncovering the purine biosynthesis pathway as crucial for therapeutic efficacy. ME-344 induced a dose-dependent decrease in the oxygen consumption rate (by Seahorse assay), in both AraC-sensitive and -resistant AML cell lines, and in R/R AML patient samples, which was further significantly potentiated by combination with VEN.Finally, in an aggressive AML xenograft model, ME-344 (200 mpk, i.v.) combined with subtherapeutic doses of VEN (25 mpk) reduced circulating leukemia burden and extended survival. Ongoing in vivo studies in AML PDX models will address the impact of ME-344 in the context of acquired AraC- and Bcl-2- resistance. In summary, our findings indicate ME-344 alone or in combination with Bcl-2 inhibition constitutes an important therapeutic modality that targets a unique metabolic vulnerability of AML. Citation Format: Katie H. Hurrish, Yongwei Su, Shraddha Patel, Sandra E. Wiley, Zhanjun Hou, Jenna Carter, Hasini Kalpage, Maik Hüttemann, Connie Weng, Holly Edwards, Lisa Polin, Jing Li, Jay Yang, Larry H. Matherly, Sergej Naumovich Konoplev, Jeffrey W. Taub, Marina Konopleva, Yubin Ge, Natalia Baran. ME-344, a novel isoflavone mitochondrial inhibitor, in combination with venetoclax constitutes a new metabolism-targeted approach to overcome resistance to Bcl-2 inhibition and standard of care treatment in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3785.

Abstract Mutations in KRAS at G12, G13, and Q61 are oncogenic drivers in many cancers, including lung, colorectal, pancreatic, multiple myeloma, and uterine carcinomas. KRAS mutations are frequently accompanied by stabilization of the MYC oncoprotein through increased MYC transcription and decreased protein degradation that is mediated by phosphorylation of MYC on Ser 62 by ERK and CDK9 kinases. Voruciclib is a novel oral inhibitor of CDKs 9, 4, 6, and 1 that is currently being tested in Phase 1B clinical trials for B-cell malignancies and acute myeloid leukemia. Voruciclib inhibition of CDK9 leads to decreased expression of transcriptional targets of RNA Pol II, such as Mcl1 and MYC. To test whether voruciclib could be effective in cancers driven by dysregulated KRAS-MYC signaling, ~20 cancer cell lines with KRAS mutations (G12A, G12C, G12D, G12S, G12V, G13C, G13D, Q61H) were treated in preclinical studies with voruciclib. MTS and Cell Titer Glo assays were used to monitor growth in vitro. Voruciclib decreased viability in all cell lines tested. To investigate MYC protein stability, MIA PACA-2 (G12C) cells were treated with 4 µM voruciclib for 5-240 min, followed by SDS-PAGE and Western Blotting analysis with α-MYC and α-pSer62-MYC antibodies. Voruciclib treatment resulted in a reduction in phosphorylation of MYC on Ser 62. A 60% decrease in pSer62 was observed after 5 min that reached 80% by 60 min. In contrast, there was no decrease in total MYC protein at either 5 or 15 min. A 10% reduction in total MYC was observed at 60 min that reached 50% at 240 min. The ability of voruciclib to inhibit tumor growth in vivo was also tested in murine xenograft models. KRAS mutant human cancer cells HCT-116 (CRC, KRAS G13D), SW-480 (CRC, KRAS G12V), and H-460 (NSCLC, KRAS Q61H) were injected subcutaneously into SCID mice. Once tumors reached 5-10 mm in diameter, voruciclib or vehicle were administered orally at 50, 100, or 200 mg/kg OD for 11-14 days. Tumors were measured every 2-3 days, and growth inhibition relative to control was calculated. Significant tumor growth inhibition (>50%) was observed at all doses of voruciclib tested. Collectively, these data demonstrate that voruciclib inhibition of CDK9 leads to reduced phosphorylation of MYC on Ser 62 followed by a decrease in total MYC protein in MIA PACA-2 cells and inhibition of growth in multiple KRAS mutant cancer cell lines in vivo and in vitro. This suggests that voruciclib could be an attractive therapeutic option for cancers driven by KRAS-MYC. Citation Format: Sandra E. Wiley, Yongwei Su, Yubin Ge. Voruciclib, a CDK9 inhibitor, downregulates MYC and inhibits proliferation of KRAS mutant cancers in preclinical models [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 1962.

The 5-year survival rate for adult patients with acute myeloid leukemia (AML) treated with cytarabine-based chemotherapy remains less than 30%, due to drug resistance and disease relapse. Recently, a selective inhibitor of anti-apoptotic Bcl-2, venetoclax, was approved by the FDA in combination with low dose cytarabine or hypomethylating agents for treating newly diagnosed AML patients who are 75 years of age or older or for those who are unfit for standard chemotherapy, providing more treatment options for this group of patients. Although the response rate to these newly approved combination therapies is reported to be 70%, the median overall survival is only 10-18 months showing that the duration of response is limited. Therefore, novel therapeutic agents are in demand to enhance venetoclax activity against AML and to combat AML resistant to cytarabine-based chemotherapy.

The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.

Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.

Ca2+ signaling is important for many cellular and physiological processes, including cardiac function. Although sarcoplasmic reticulum (SR) proteins involved in Ca2+ signaling have been shown to be phosphorylated, the biochemical and physiological roles of protein phosphorylation within the lumen of the SR remain essentially uncharacterized. Our laboratory recently identified an atypical protein kinase, Fam20C, which is uniquely localized to the secretory pathway lumen. Here, we show that Fam20C phosphorylates several SR proteins involved in Ca2+ signaling, including calsequestrin2 and Stim1, whose biochemical activities are dramatically regulated by Fam20C mediated phosphorylation. Notably, phosphorylation of Stim1 by Fam20C enhances Stim1 activation and store-operated Ca2+ entry. Physiologically, mice with Fam20c ablated in cardiomyocytes develop heart failure following either aging or induced pressure overload. We extended these observations to show that non-muscle cells lacking Fam20C display altered ER Ca2+ signaling. Overall, we show that Fam20C plays an overarching role in ER/SR Ca2+ homeostasis and cardiac pathophysiology.

Phosphoproteomics studies have reported phosphorylation at multiple sites within collagen, raising the possibility that these post-translational modifications regulate the physical or biological properties of collagen. In this study, molecular dynamics simulations and experimental studies were carried out on model peptides to establish foundational principles of phosphorylation of Ser residues in collagen. A (Gly-Xaa-Yaa)(11) peptide was designed to include a Ser-containing sequence from type I collagen that was reported to be phosphorylated. The physiological kinase involved in collagen phosphorylation is not known. In vitro studies showed that a model kinase ERK1 (extracellular signal-regulated protein kinase 1) would phosphorylate Ser within the consensus sequence if the collagen-like peptide is in the denatured state but not in the triple-helical state. The peptide was not a substrate for FAM20C, a kinase present in the secretory pathway, which has been shown to phosphorylate many extracellular matrix proteins. The unfolded single chain (Gly-Xaa-Yaa)(11) peptide containing phosphoSer was able to refold to form a stable triple helix but at a reduced folding rate and with a small decrease in thermal stability relative to the nonphosphorylated peptide at neutral pH. These biophysical studies on model peptides provide a basis for investigations into the physiological consequences of collagen phosphorylation and the application of phosphorylation to regulate the properties of collagen biomaterials.

Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores. Histidine-rich Ca-binding protein (HRC) resides in the lumen of the SR, where it contributes to the regulation of Ca cycling by protecting stressed or failing hearts. The common Ser96Ala human genetic variant of HRC strongly correlates with life-threatening ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. However, the underlying molecular pathways of this disease remain undefined. Here, we demonstrate that family with sequence similarity 20C (Fam20C), a recently characterized protein kinase in the secretory pathway, phosphorylates HRC on Ser96. HRC Ser96 phosphorylation was confirmed in cells and human hearts. Furthermore, a Ser96Asp HRC variant, which mimics constitutive phosphorylation of Ser96, diminished delayed aftercontractions in HRC null cardiac myocytes. This HRC phosphomimetic variant was also able to rescue the aftercontractions elicited by the Ser96Ala variant, demonstrating that phosphorylation of Ser96 is critical for the cardioprotective function of HRC. Phosphorylation of HRC on Ser96 regulated the interactions of HRC with both triadin and SERCA2a, suggesting a unique mechanism for regulation of SR Ca homeostasis. This demonstration of the role of Fam20C-dependent phosphorylation in heart disease will open new avenues for potential therapeutic approaches against arrhythmias.

The mechanisms and machinery regulating calcium cycling through the sarcoplasmic reticulum (SR), a secretory pathway calcium storage organelle, remain incompletely understood, and further insight is critical for discerning normal and diseased heart function. An overlooked component is luminal SR protein phosphorylation. Our lab recently identified an atypical protein kinase in the secretory pathway lumen, Fam20C, and have demonstrated its role in calcium homeostasis for proper development of bone and teeth. This novel kinase phosphorylates Ser residues within highly conserved Ser-x-Glu/pSer (SxE) motifs, and is interestingly responsible for the overwhelming majority of secretory pathway protein phosphorylation. Therefore, we hypothesize that Fam20C phosphorylates proteins in the cardiac SR lumen, and that this phosphorylation will play an important role in SR calcium cycling. Indeed, we demonstrate that Fam20C phosphorylates SR proteins that have SxE sites, including Calsequestrin 2, Triadin, Sarcalumenin, Calreticulin, and Calumenin, all of which play important roles in SR calcium handling. Furthermore, we developed a cardiac specific Fam20C knockout mouse model. Following transverse aortic constriction (TAC) induced pressure overload, we find that Fam20C ablation reduces cardiac function and shows significantly increased signs of heart failure. Also, Fam20C ablation following TAC causes reduction in key regulators of SR calcium cycling, including SR Calcium-ATPase type 2a (SERCA2a) and phospholamban. Our results establish Fam20C as a novel cardioprotective signaling molecule, and open new avenues for potential therapeutic approaches to cardiovascular disease.

Protein kinases are evolutionarily conserved enzymes that transfer a molecule of phosphate from ATP to protein substrates in a process known as phosphorylation. In 1883, the secreted milk protein casein was shown to contain phosphorous. In hindsight, this was the first indication for the existence of protein kinases that were ultimately discovered nearly one‐century later using casein as the substrate. We now know that protein phosphorylation is a universal mechanism that regulates nearly every aspect of cellular life. Despite the fact that casein was identified as the first phosphoprotein, the responsible kinase had remained obscure. The enzymes classically referred to as “casein kinases” do not mediate the physiological phosphorylation of casein because they are nuclear and cytosolic proteins and do not encounter casein within the secretory pathway. Numerous other secreted proteins and peptide hormones are phosphorylated; however, the molecular identities of the kinases responsible for these modifications were unknown as well. As a consequence, research on extracellular protein phosphorylation has been largely undeveloped. We discovered a novel family of atypical secretory pathway kinases that phosphorylate secreted proteins. This new kinase family is so different from canonical kinases that it was not included as a branch on the human “kinome” tree. One member of this new family, Fam20C, is the Golgi casein kinase, an enzyme that escaped identification for many years. Fam20C contains a signal peptide that directs it to the lumen of the secretory pathway where it phosphorylates proteins on S‐x‐E/pS motifs. The importance of this discovery is highlighted by the fact that ~75% of human serum, plasma and cerebrospinal fluid phosphoproteins are phosphorylated within this motif. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing and mass spectrometry, we identify more than 100 phosphoproteins as Fam20C substrates. Functional annotations of Fam20C substrates suggest roles for the kinase in a broad spectrum of biological processes, including cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of life. Several secreted proteins are phosphorylated, but the kinases responsible are unknown. We identified a family of atypical protein kinases which have N-terminal signal sequence and localize to the lumen of the endoplasmic reticulum and the Golgi apparatus. One of these kinases known as Fam20C is the Golgi casein kinase and it phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and many other secretory proteins including the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) implicated in biomineralization. Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome. Another family member, Fam20B, is a xylose kinase important in the maturation of proteoglycans. The function of other FAM 20C family members will also be addressed.

The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

BackgroundTargeting the mitochondria during ischemia/reperfusion (IR) can confer cardioprotection leading to improved clinical outcomes. The cardioprotective potential of (−)-epicatechin (EPI) during IR via modulation of mitochondrial function was evaluated.Methods and resultsIschemia was induced in rats via a 45min occlusion of the left anterior descending coronary artery followed by 1h, 48h, or 3week reperfusion. EPI (10mg/kg) was administered IV 15min prior to reperfusion for the single dose group and again 12h later for the double dose group. Controls received water. Experiments also utilized cultured neonatal rat ventricular myocytes (NRVM) and myoblasts. A single dose of EPI reduced infarct size by 27% at 48h and 28% at 3week. Double dose treatment further decreased infarct size by 80% at 48h, and 52% by 3weeks. The protective effect of EPI on mitochondrial function was evident after 1h of reperfusion when mitochondria demonstrated less respiratory inhibition, lower mitochondrial Ca2+ load, and a preserved pool of NADH that correlated with higher tissue ATP levels. Mechanistic studies in NRVM revealed that EPI acutely stimulated maximal rates of respiration, an effect that was blocked by inhibitors of the mitochondrial pyruvate carrier, nitric oxide synthase, or soluble guanylyl cyclase. In myoblasts, knockdown of components of the mitochondrial pyruvate carrier blocked EPI-induced respiratory stimulation.ConclusionsIV EPI confers cardioprotection via preservation of mitochondrial function potentially through enhanced substrate provision. These provocative results document a novel mechanism of a natural product with potential clinical utility.

The family with sequence similarity 20, member C (Fam20C) has recently been identified as the Golgi casein kinase. Fam20C phosphorylates secreted proteins on Ser-x-Glu/pSer motifs and loss-of-function mutations in the kinase cause Raine syndrome, an often-fatal osteosclerotic bone dysplasia. Fam20C is potentially an upstream regulator of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), because humans with FAM20C mutations and Fam20C KO mice develop hypophosphatemia due to an increase in full-length, biologically active FGF23. However, the mechanism by which Fam20C regulates FGF23 is unknown. Here we show that Fam20C directly phosphorylates FGF23 on Ser(180), within the FGF23 R(176)XXR(179)/S(180)AE subtilisin-like proprotein convertase motif. This phosphorylation event inhibits O-glycosylation of FGF23 by polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3), and promotes FGF23 cleavage and inactivation by the subtilisin-like proprotein convertase furin. Collectively, our results provide a molecular mechanism by which FGF23 is dynamically regulated by phosphorylation, glycosylation, and proteolysis. Furthermore, our findings suggest that cross-talk between phosphorylation and O-glycosylation of proteins in the secretory pathway may be an important mechanism by which secreted proteins are regulated.