Supplementary Tables S1-S4 and Figures S1-S3. Tables S1/S2: % tumor response in mice with HCT116 and A2058 tumor xenografts following treatment; Table S3: Diagram showing dose-decision guidelines utilized in the MK-2206 clinical study based on observed toxicities; Table S4: number of patients with drug-related AEs in all courses of treatment; Figure S1/S2: Line graph showing the relative tumor volume and body weight observed in patients over time expressed in terms of days following treatment; Figure S3: Patient presenting with Grade III rash

Abstract Purpose: KRAS is the most commonly mutated oncogene in human tumors. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. Experimental Design: We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumors. Recommended dosing schedules were defined as MK-2206 at 135 mg weekly and selumetinib at 100 mg once daily. Results: Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug–drug interactions. Clinical antitumor activity included RECIST 1.0–confirmed partial responses in non–small cell lung cancer and low-grade ovarian carcinoma. Conclusion: Responses in KRAS-mutant cancers were generally durable. Clinical cotargeting of MEK and AKT signaling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). Clin Cancer Res; 21(4); 739–48. ©2014 AACR.

G Protein-Coupled Receptor Kinase 2 (Grk2) is a serine-threonine kinase widely expressed in the human body. The GRK family of kinases is known for its role in GPCR phosphorylation and desensitization upon GPCR activation by agonists. Besides its role in GPCR phosphorylation, Grk2 has been shown to phosphorylate non-GPCR receptors and cytoplasmic substrates and has also been demonstrated to play phosphorylation-independent roles in cells. Using pooled CRISPR screening we identified that Grk2 knockout inhibits pancreatic and bladder cancer cell line proliferation in vitro and tumor growth in xenografts in vivo, which has not been demonstrated before. Rescue experiments re-expressing wildtype or kinase inactive Grk2 showed that this effect is dependent on catalytically active Grk2. We developed Grk2 inhibitors with exquisite enzymatic potency and anti-proliferative activity in pancreatic cancer cell lines. On a cellular level Grk2 knockout and Grk2 inhibitors slow down G2/M cell cycle progression in pancreatic cancer cell lines, and Grk2 inhibitor CYG-N-2278 induced a 71% tumor growth inhibition in PAXF1657 pancreatic PDX-derived xenografts. Besides its direct role in cancer cell growth Grk2 also plays a role in immune cell function. Grk2 is highly expressed in immune cells, and we have demonstrated that Grk2 inhibitors upregulate proinflammatory cytokine expression in myeloid cells. In the MC38 colorectal syngeneic tumor model implanted into immunocompetent C57BL6 mice CYG-N-2278 Grk2 inhibitor resulted in a 39% tumor growth inhibition, whereas no effect was observed on MC38 tumor growth in immunodeficient NSG mice, suggesting an immune cell mediated tumor growth inhibition. Analysis of The Cancer Genome Atlas data has showed that Grk2 is amplified across multiple tumor types, including bladder, uterine and cholangiocarcinoma cancer, and that Grk2 amplification is prognostic for tumor outcome, confirming the human relevance and suggesting a translatable patient selection strategy. In conclusion, we identified Grk2 as novel target for oncology, demonstrating a direct effect of Grk2 inhibition on cancer cells through inhibiting G2/M cell cycle progression and an indirect effect on tumor growth through immune cell activation. Cygnal’s Grk2 inhibitors possess superior potency over previous Grk2 inhibitors and have shown promising preclinical anti-tumor efficacy. Citation Format: Alexandra Lantermann, Eugene Chekler, Bruce Lefker, Garmen Yuen, Vanessa Lam, Matthew Strickland, Justyne Pennacchio, Aaron Fulgham, Dara Bree, Thomas Perekslis, Grazia Piizzi, Hongyue Dai, Tim Zheng, John Wagner, Pearl Huang. Identification of Grk2 as novel oncology target and development of potent Grk2 inhibitors [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 166.

The nervous system plays an important role in human health and disease, and the unique morphologies of the neurons underlie its ability to interface with tissues and organs throughout the entire body. In vitro, neurons can be grown alone or with other cell types to gain insight into how they communicate with other cell types in a more controlled experimental setup. To measure neuron growth and to study neuronal connectivity in vitro, neurite identification is an essential readout. However, non-specific binding of fluorescence probes, a fundamental issue of fluorescence imaging, impairs neurite identification through conventional mathematical morphology-based methods, especially in neuron and other cell type co-culture imaging conditions. Here, we utilized a deep learning algorithm and developed a computational tool called DeepNeurite (TM), to overcome this challenge. We demonstrated that DeepNeurite (TM) can accurately identify neurite structure in images acquired from microfluidic compartmentalized chambers where neurons were co-cultured, such as with a human prostate cancer cell line, PC3. We further validated that the model can be generalized to handle a direct co-culture in which neurons and lung cancer cells (DMS273) are grown intermingled in the same well. Using this method, we observed more neurite growth into PC3 containing chambers in microfluidic compartmentalized chambers, which could be blocked by an NGF antibody. Finally, we applied DeepNeurite (TM) coupled with functional calcium imaging to study the communication of primary sensory neurons and cancer cells. We showed that the cancer cells closer to neurites exhibit greater calcium activity in response to neuronal stimulation. This method opens lots of opportunities to study the effect of neurons on various other cell types. This model could further tackle the off-target labeling of the fluorescence probe in other subcellular structures or cell types.

A hallmark of endometriosis - a chronic debilitating condition whose causes are poorly understood - is neuronal innervation of lesions. Recent evidence demonstrates that the peripheral nervous system plays an important role in the pathophysiology of this disease. Sensory nerves, which surround and innervate endometriotic lesions, not only drive the chronic and debilitating pain associated with endometriosis but also contribute to a pro-growth phenotype by secreting neurotrophic factors and interacting with surrounding immune cells. The diverse array of contributions that neurons play in endometriosis indicate that it should be considered as a nerve-centric disease. This review is focused on the emerging field of exoneural biology and how it applies to the field of endometriosis, in particular the role that peripheral nerves play in driving and maintaining endometriotic lesions. A better understanding of the mechanisms of neuronal contribution to endometriosis, as well as their interactions with accompanying stromal and immune cells, will unearth novel disease-relevant pathways and targets, providing additional, more selective therapeutic horizons.

Abstract Linking features of the tumor microenvironment (TME) to patient outcome is ambitious yet crucial to understand cancer progression and prognosis in order to develop new therapies. The current method to assess histopathological images is visual inspection by a professional, generally a pathologist. While this is an effective approach for diagnosis, it does not scale well for discovery biology and population-based studies for drug discovery purposes. An automated approach would allow for a more rapid, systematic, and comprehensive analysis of several morphological features of the TME by taking advantage of thousands of already existing slides within databases and biobanks. Recent studies have shown intriguing relationships between innervation in tumors (tumor exoneural biology) and patient outcomes. Here we present an automated tool that can detect and quantify nerve presence in tumors. We manually annotated a set of digital slides from The Cancer Genome Atlas (TCGA) in order to develop a deep learning model to quantify the presence of nerves in head and neck tumors stained with H&E. This tool is generalizable and was applied to identify patterns in the appearance of tumor-infiltrating lymphocytes (TILs) in and around tumors. It may be further applied to other structural features such as blood vessels in order to characterize and correlate these features within the TME in hundreds of images across cancer types. This enables integration of imaging features with multi-omics data to uncover potential biological pathways that are upregulated in groups with dense innervation compared to sparse innervation in cancer. The main advantage of this approach is the ability to utilize many public databases in which the features of interest can be correlated with reported patient comorbidities, treatments, and phenotypes. This platform-based methodology can be expanded to other disease areas and could ultimately provide valuable insight about exoneural biology and its role in disease physiology to identify new avenues for therapies. Citation Format: Alison R. Miller, Daniel Krasnonosenkikh, Monica Thanawala, Kai Chih Huang, George V. Thomas, Alexandra B. Lantermann, Hongyue Dai, Masoud Sadaghiani, John A. Wagner, Pearl Huang. Automated nerve identification in histopathology slides enables comprehensive analysis of innervation in cancer and tumor neurobiology [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P262.

Although the role of nerves in stimulating cellular growth and dissemination has long been described in tissue regeneration studies, until recently a similar trophic role of nerves in disease was not well recognized. However, recent studies in oncology have demonstrated that the growth and dissemination of cancers also requires the infiltration of nerves in the tumor microenvironment. Nerves generate various neurosignaling pathways, which orchestrate cancer initiation, progression, and metastases. Similarly, nerves are increasingly implicated for their regulatory functions in immunity and inflammation. This orchestrator role of nerves in cellular and molecular interactions during regeneration, cancer, immunity, and inflammation offers new possibilities for targeting or enhancing neurosignaling in human health and diseases.

Transcriptomics and sequencing analyses of tumors from patients provide invaluable information about the cells present in the tumor microenvironment (TME); however, nerves, whose cell bodies are absent from most peripheral TMEs, have eluded such approaches. Recent emerging data highlight the functional importance of innervation in the TME and its contribution to tumor progression, metastasis, and treatment resistance. The Exoneural Platform developed at Cygnal Therapeutics allows for investigation of complex biology in the context of multi-cell culture conditions in vitro and in vivo. As part of this platform, we have developed tools for rapid and selective manipulation of different cell types with the ability to monitor multiple cell specific read-outs. Here, we used a combination of approaches including RNA-seq, bioinformatics, immunohistochemistry, and microscopy to investigate the role of synaptic function in cancer biology. Transcriptomics analyses on co-cultures revealed a direct interaction between nerves and cancer cells based on cell type specific gene expression and ligand - receptor interaction assessment. Both neurons and cancer cells showed a profound shift in their gene expression profile when cultured together. This differential gene expression was reversed if neurons were ablated, pointing to the reversibility of this change and, potentially, the disease state itself. Examination for presence and function of synaptic proteins showed that synaptic proteins, such as PSD95 and synapsin1, were expressed in co-cultures, and in many cases, a co-localization of pre - and post - synaptic markers was observed. Selective stimulation of dorsal ganglion root (DRG) neurons, co-cultured with cancer cells, resulted in a robust and acute increase of cytosolic calcium in cancer cells. Bioinformatic image analysis confirmed that this calcium influx in cancer cells directly correlated with proximity and density of neurites to cancer cells. A calcium response was not observed if DRGs and cancer cells were cultured in two separate chambers between which media can freely travel, confirming that close proximity is required for this interaction. Pharmacological and genetic knock down of key synaptic proteins resulted in changes in cancer cell calcium influx and affected proliferation and gene expression. Bioinformatics analyses, based on public and proprietary data, have identified the synaptic pathway as a major contributing node that influences cancer cell biology in the context of several cancer types. These data collectively point to the importance of exoneural biology, and more specifically, synaptic biology in cancer. More importantly, these results are likely to suggest new oncology targets that have not been identified or pursued previously. Citation Format: Monica Thanawala, Chih-Chieh Wang, Jesse G. Turner, Kai-Chih Huang, Lexiang Ji, Alison Miller, Alexandria Fink, Shan Lou, Alexandra B. Lantermann, Hongyue Dai, John A. Wagner, Grazia Piizi, Jonathan B. Hurov, Pearl Huang, Amir M. Sadaghiani. Neural communication to peripheral tumors regulates cancer cell activity [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 1450.

Tumor innervation has recently been documented and characterized in various settings and tumor types. However, the role that nerves innervating tumors play in the pathogenesis of cancer has not been clarified. In this study, we searched for neural signaling from bulk RNA sequencing from The Cancer Genome Atlas (TCGA) dataset and looked for patterns of interactions between different cell types within the tumor environment. Using a presynapse signature (PSS) as a probe, we showed that multiple stromal cell types crosstalk and/or contribute to neural signals. Based on the correlation and linear regression, we hypothesized that neural signals contribute to an immune-suppressive tumor microenvironment (TME). To test this hypothesis, we performed in vitro dorsal root ganglion (DRG)/macrophage coculture experiments. Compared to the M2 macrophage monoculture, the DRG/M2 macrophage coculture prevented anti-inflammatory M2 to pro-inflammatory M1 polarization by LPS stimulation. Finally, a survey of different TCGA tumor types indicated that higher RNA neural signature is predictive of poor patient outcomes in multiple tumor types.

Abstract Background Cygnal Therapeutics, pioneer in exoneuronal biology, has identified the neuronal protein, Neuropilin-1 (NRP1) as a critical regulator of tumor growth and anti-tumor immunity. NRP1 is a co-receptor that complexes with diverse ligands and their cognate receptors. As such, it plays a role in multiple different biological processes, including axon guidance and angiogenesis. NRP1 contains two CUB domains (a1 and a2) involved in binding the ligand Semaphorin3A (SEMA3A), two Factor V/VIII domains (b1 and b2) involved in VEGF ligand binding and one MAM domain (c domain). While functional antibodies with anti-tumor activity have been generated against the SEMA3A and VEGF binding domains, targeting other domains of NRP1 has not been well-studied. For example, the c-domain of NRP1 has been implicated in the dimerization of NRP1, a prerequisite for functionality. Thus c-domain blockers might inhibit multiple signaling pathways required for tumor growth. Alternatively, binders targeting the interface of two different domains might allow for simultaneous blocking of multiple ligands. We therefore hypothesized that these novel binders to alternative regions of NRP1 such as the b1/b2 interface and the c-domain would have improved and significant anti-tumor activity. Methods Antibodies with diverse binding characteristics were tested for in vivo anti-tumor activity in multiple syngeneic models, including anti-PD1 non-responsive models. Ability of these of the anti-NRP1 antibodies to elicit an adaptive immune response was evaluated. Results Novel binders to alternative regions of NRP1 such as domain interfaces and the c-domain resulted in significant tumor growth inhibition in multiple syngeneic models. Additionally, the NRP1 antagonists induced a robust CD8 T cell response, indicating immune-stimulatory role of these antibodies. Conclusions Cygnal has developed fully human monoclonal antibodies against previously untargeted domains of NRP1. These novel domain binders show remarkable inhibition of tumor growth in multiple syngeneic models and offer a novel means of therapeutic intervention in patients. Citation Format: Shalini Sethumadhavan, Eric Zhu, Aaron Fulgham, Mandana Abbassi, Ameya Apte, Tiffany Liao, Chengfeng Merriman, Katherine Molloy, Caitlin Stein, Amber Hanna, James Geoghegan, Bianka Prinz, Hongyue Dai, Jonathan Hurov, Pearl Huang, Daniel Blom. Novel mechanisms of Neuropilin-1 inhibition result in improved tumor growth inhibition in vivo [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 526.

Background Neuropilin-1 (NRP1) is a co-receptor that complexes with diverse ligands and their cognate receptors. As such, it plays a role in multiple different biological processes, including axon guidance and angiogenesis. NRP1 contains two CUB domains (a1 and a2) involved in binding the ligand Semaphorin3A (SEMA3A), two Factor V/VIII domains (b1 and b2) involved in VEGF ligand binding and one MAM domain (c domain). While functional antibodies with anti-tumor activity have been generated against the SEMA3A and VEGF binding domains, little attention has been paid to the c domain of NRP1, which has been implicated in the dimerization of NRP1, a prerequisite for functionality. We therefore hypothesized that c domain-binding antibodies would offer an opportunity to generate functional inhibitors of both SEMA3A and VEGF signaling and therefore improved anti-tumor activity. Methods Recombinant human NRP1 comprising all subdomains was used to identify fully human anti-NRP1 antibodies. Specific antibodies were tested for their ability to block NRP1 interactions with recombinant SEMA3A and VEGF protein in vitro. Blocking antibodies were subsequently assessed for their functional effects, such as inhibition of SEMA3A-mediated growth cone collapse. Antibodies with diverse binding characteristics were then tested for in vivo anti-tumor activity in multiple cancer models of interest. Results Recombinant NRP1 containing the a1, a2, b1, b2 and c subdomains was used to successfully identify a series of specific monoclonal antibodies that cross-reacted with Cynomolgus monkey and mouse NRP1, but not human NRP2. Except for the a2 domain, epitope mapping showed an even distribution of mAbs for binding to each of the NRP1 subdomains, including the c domain that has been proposed to play a role in dimerization. Using biolayer interferometry, we identified antibody classes with direct SEMA3A and/or VEGF blocking properties. Further optimization of these antibodies yielded mAbs with subnanomolar affinities that showed significant tumor growth inhibition in multiple mouse models, including anti-PD1 non-responsive models. Conclusions Here we demonstrate the identification of fully human monoclonal antibodies that specifically bind to the c domain of human NRP1. A subset of these c domain binders do not block either SEMA3A or VEGF binding to NRP1 but do show in vivo efficacy, suggesting a role for the c domain of NRP1 in the formation of functional (dimeric) complexes. Thus, c domain binding antibodies show remarkable inhibition of tumor growth in mouse cancer models and offer a novel means of therapeutic intervention in patients who are refractory to immune checkpoint inhibition.

AbstractPurpose:KRAS is mutated in the majority of pancreatic ductal adenocarcinoma. MAPK and PI3K-AKT are primary KRAS effector pathways, but combined MAPK and PI3K inhibition has not been demonstrated to be clinically effective to date. We explore the resistance mechanisms uniquely employed by malignant cells.Experimental Design:We evaluated the expression and activation of receptor tyrosine kinases in response to combined MEK and AKT inhibition in KPC mice and pancreatic ductal organoids. In addition, we sought to determine the therapeutic efficacy of targeting resistance pathways induced by MEK and AKT inhibition in order to identify malignant-specific vulnerabilities.Results:Combined MEK and AKT inhibition modestly extended the survival of KPC mice and increased Egfr and ErbB2 phosphorylation levels. Tumor organoids, but not their normal counterparts, exhibited elevated phosphorylation of ERBB2 and ERBB3 after MEK and AKT blockade. A pan-ERBB inhibitor synergized with MEK and AKT blockade in human PDA organoids, whereas this was not observed for the EGFR inhibitor erlotinib. Combined MEK and ERBB inhibitor treatment of human organoid orthotopic xenografts was sufficient to cause tumor regression in short-term intervention studies.Conclusions:Analyses of normal and tumor pancreatic organoids revealed the importance of ERBB activation during MEK and AKT blockade primarily in the malignant cultures. The lack of ERBB hyperactivation in normal organoids suggests a larger therapeutic index. In our models, pan-ERBB inhibition was synergistic with dual inhibition of MEK and AKT, and the combination of a pan-ERBB inhibitor with MEK antagonists showed the highest activity both in vitro and in vivo.

Abstract Peripheral nerves were first described as a component of tumors in the late 19th century. In the mid-20th century early preclinical studies indicated that tumor cells could recruit innervation from spinal cord ganglia and that stress promotes tumorigenesis. Within the last 10 years improvements in technologies allowing neuronal tracking and regulation of neuronal function have uncovered a role for both autonomic and sensory innervation in multiple tumor types. Initial studies have implicated this biology in prostate, pancreatic, gastric and breast cancer (among others) and point to a role for nerves in initiation, maintenance and metastasis of tumors. The stimulation of tumor cell growth by neural growth factors, the recruitment of neurites by tumor cells, the invasion and migration of tumor cells along nerves (perineural invasion), and the role of innervation in driving tumor angiogenesis are just a few examples of how peripheral nerves interact with the tumor microenvironment. Indeed, an argument could be made that innervation of tumors should be considered a hallmark of cancer. We describe here a biological platform that can define and decode the role of neural signaling in cancer. This platform, which we call Exoneural Medicines Platform ™, has 6 technical components: 1) co-culture models of primary neurons with both tumor and immune cells, 2) advanced imaging modalities to define the neural component of tumors, 3) AAV and transgenic tools that allow regulation of neurons proximal to tumors in vivo and in vitro, 4) neural-focused functional genomics, 5) neural-focused bioinformatics, and 6) a neuropharmacopeia compound library for probing relevant biological pathways and identification of advanced starting points for drug discovery. We show here how the platform is being employed to characterize this exciting new space in cancer biology and to identify new drivers of disease. Citation Format: Shan Lou, Alexandria Fink, Jay Wang, Jesse Turner, Monica Thanawala, Garmen Yuen, Alexandra Lantermann, Jenny Shu, Hongyue Dai, Pearl Huang, Jonathan Hurov. Uncovering novel cancer therapeutics using the Exoneural Medicines Platform: The role of innervation in cancer [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 LB-B17. doi:10.1158/1535-7163.TARG-19-LB-B17

The pancreas is highly innervated with sensory, autonomic and enteric neurons, and recent studies have highlighted the crosstalk between neurons and pancreatic cells and their potential role in driving tumorigenesis and promoting metastasis. Published data has shown that ablation of sensory neurons in models of pancreatic ductal adenocarcinoma (PDAC) resulted in delayed tumor initiation and progression. In addition, perineural invasion (PNI), the invasion of tumor cells along the nerve, is a pathological characteristic frequently observed in PDAC. PNI has been detected in early stages of pancreatic cancer and associated with a poor outcome. We hypothesized that PDAC tumor cells express neuronal genes which may be playing a role in enabling tumor-nerve interactions and promoting PDAC tumorigenesis. Here we used pooled sgRNA CRISPR screening to probe a library of 5000 sgRNAs targeting 800 neuronal genes. We selected three established (MiaPaca-2, Panc-1 and BxPC3) and two patient derived human PDAC cell lines (PAXF1657 and PAXF1997) and performed in vitro and in vivo CRISPR screens. sgRNAs targeting the CYFIP1 gene were negatively selected across multiple cell line models in vitro and in vivo. Cytoplasmic FMR1 interacting protein 1 (CYFIP1) is a protein that has been described to regulate actin polymerization and protein translation through two distinct protein complexes. CYFIP1 is a member of the heteropentameric WAVE regulatory complex (WRC) which includes CYFIP1, WAVE, NAP1, ABI, and HSPC300. Rac1 binding to CYFIP1 results in activation of WRC, allowing WAVE to interact with Apr2/3 and enabling actin polymerization. In addition, CYFIP1 can interact with fragile X mental retardation protein (FMRP) and eIF4E, acting as a translation repressor. CYFIP1 is highly expressed at excitatory synapses of neurons and is required for proper dendritic spine morphology. CYFIP1 has been linked to neurological and neuropsychiatric conditions such as autism, epilepsy and schizophrenia due to its location in a chromosomal region (15q11.2) with frequent copy number variations. In order to validate the pooled CRISPR screen results we individually knocked out CYFIP1 in PDAC cancer cell lines. We were able to show that knockout of CYFIP1 reduced proliferation of PDAC cells in vitro and dramatically reduced tumor growth in vivo. Additionally, knockout of CYFIP1 resulted in reduced protein levels of WRC members WAVE1 and NCKAP1, suggesting destabilization of the complex. The mechanism by which CYFIP1 promotes tumor growth has yet to be elucidated, but our data shows that CYFIP1 is playing a critical role in PDAC tumorigenesis and is a potential therapeutic target. Citation Format: Kiley Couto, Matthew Strickland, Tiffany Liao, Mortada Najem, Aaron Fulgham, Ameya Apte, Pearl Huang, Jonathan Hurov, Alexandra Lantermann. The autism and schizophrenia-associated CYFIP1 protein is required for pancreatic tumor growth and presents a potential therapeutic target [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 LB-C05. doi:10.1158/1535-7163.TARG-19-LB-C05

Purpose: KRAS is the most commonly mutated oncogene in human tumors. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. Experimental Design: We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumors. Recommended dosing schedules were defined as MK-2206 at 135 mg weekly and selumetinib at 100 mg once daily. Results: Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug–drug interactions. Clinical antitumor activity included RECIST 1.0–confirmed partial responses in non–small cell lung cancer and low-grade ovarian carcinoma. Conclusion: Responses in KRAS-mutant cancers were generally durable. Clinical cotargeting of MEK and AKT signaling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). Clin Cancer Res; 21(4); 739–48. ©2014 AACR.

There has been increasing interest in developing cancer therapies targeting PI3K pathway nodes. However, inhibition of a single node in PI3K pathway such as mTOR by rapamycin analogues results in compensatory activation of survival signaling pathway such as AKT and thereby limiting monotherapy activity. Therefore, in most tumor types, multipathway inhibition, guided by an in-depth understanding of feedback and crosstalk between pathways, may be required for tumor regression. It is well known that that mTOR inhibition upregulates pAKT via the S6K-IRS2 negative feedback loop. Inhibiting both mTOR and IGF1R may therefore ablate such feedback upregulation and lead to clinical response. However, it is unclear which tumor types will respond to the combination in the clinic. Here, we present preclinical and molecular profiling data that supports LumB breast cancer as a potential indication for MK-8669/MK-0646 (mTOR/IGF1 R inhibitor combination). When a panel of over 60 breast cancer cell lines was treated with MK-8669, ER+ cell lines and HER2+ cell lines were clearly more responsive than the triple negative ones. Meanwhile, the status of the MAPK pathway, as captured by a RAS gene expression signature, correlated with resistance to MK-8669 across the panel. Moreover, MK-8669 treatment unregulated multiple key nodes of the IGF1R pathway in the breast cell line panel, especially IRS2, whose upregulation correlated with the response to MK-8669. Furthermore, IGF1R, whose mRNA level correlates with the response to MK-0646 (IGF1 Ri) in multiple internal and external studies, is the highest in a subset of LumB tumors, suggesting a potential dependency on the IGF1R pathway in those tumors. Finally, a comparison between the gene expression profiles of breast cancer cell lines and tumors revealed that the ER+ breast cancer cell lines only represented the LumB, not LumA, subtype of human breast tumors. Taken together, these findings allow us to hypothesize that LumB breast cancer may be enriched in responders to the MK-0646/MK-8669 combination. In 2009, Merck initiated a phase I trial of MK-8669/MK-0646 in which clinical response was observed in LumB breast cancer patients. A phase II clinical trial has been initiated to test the hypothesis retrospectively. This talk is also presented as Poster A52. Citation Information: Clin Cancer Res 2010;16(14 Suppl):PR3.

Abstract In order to facilitate the development of HGF/ c-MET inhibitors, we undertook a genome-wide analysis to identify a “c-MET activation signature” that could be used to identify tumors that are potentially dependent on HGF / c-MET signaling and therefore more responsive to HGF/ c-MET inhibition. We first developed a c-MET co-expression signature by identifying genes that correlate with the mRNA expression of c-MET across internal panels of kidney, lung, and colorectal tumors. Many genes correlated with the expression of c-MET, including the epidermal growth factor receptor (EGFR). In order to assess the biology of genes co-expressed with cMET, we performed pathway enrichment analysis to identify pathways or pre-existing signatures that were enriched in this cMET co-expression signature more than we would expect by chance. Our previously reported RAS signature (Loboda et al, 2009 AACR annual meeting) was the top pathway enrichment, indicating a link between genes involved in RAS signaling and cMET expression across tumors. To further assess this relationship, we tested the correlation between c-MET and our RAS signature across independent cohorts of breast and lung tumors, including primary and metastatic samples. A strong correlation between c-MET expression and our RAS signature was present in all cases and was conserved in metastatic tumors. We then assessed the relationship between c-MET and RAS activation using reverse-phase protein arrays measuring approximately 70 proteins across a panel of 89 lung cancer cell lines. Four proteins were identified that significantly correlated positively with the RAS signature: total ERBB4, pMEK, pERK, and pMET. The finding that pMET correlated with RAS signature in addition to pMEK and pERK supports the connection between c-MET signaling and RAS activation, as this relationship is observed on the phospho-protein as well as mRNA levels. Taken together, these data suggest that cMET can serve as a driver of RAS signaling. To test this, we performed a xenograft study in which GTL-16 xenografts (MET amplified gastric cancer cell line) were treated with a novel small molecule inhibitor of c-MET (MK-8033). Animals were treated with vehicle or with a single dose of 10, 33, 66, 100, or 200 mpk MK-8033. At each dose, we collected samples for profiling at 2, 8, and 12 hours post-dose. MK-8033 treatment caused a dose-and time-dependent inhibition of the RAS signature at all three time points, and the level of RAS signature inhibition was related to efficacy. These data support the hypothesis that cMET is a driver of RAS signaling in some tumor contexts and suggest that basal levels of RAS pathway activation may be an important determinant of response to HGF / c-MET inhibition in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 107.

Background Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. Methods We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. Results The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. Conclusions These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.