Competing interests C.J.D. is on the Scientific Advisory Board of Mirati Therapeutics. A.C.K. has financial interests in Vescor Therapeutics, LLC. A.C.K. is an inventor on patents pertaining to KRAS-regulated metabolic pathways, redox control pathways in pancreatic cancer, targeting GOT1 as a therapeutic approach, and the autophagic control of iron metabolism. A.C.K. is on the Scientific Advisory Board of Cornerstone/Rafael Pharmaceuticals.

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRAS G12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: it is rare in lung and colorectal cancers (~1%), yet relatively common (~20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRAS G12R is functionally distinct from the more common KRAS G12D or KRAS G12V mutant proteins (KRAS G12D/V ). We found that KRAS G12D/V but not KRAS G12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRAS G12D/V but not KRAS G12R -mutant PDAC. Surprisingly, we found that KRAS G12R is defective for interaction with a key effector, p110α phosphoinositide 3-kinase (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRAS G12R -mutant PDAC. Finally, we determined that KRAS G12R -mutant PDAC displayed a distinct drug sensitivity profile compared with KRAS G12D -mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy.Hobbs et al.

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that of highly recurrent missense mutations in the GTPase RHOA. The function of these mutations has remained unresolved. We demonstrate that RHOA Y42C , the most common RHOA mutation in DGC, is a gain-offunction oncogenic mutant, and that expression of RHOA Y42C with inactivation of the canonical tumor suppressor Cdh1 induces metastatic DGC in a mouse model. Biochemically, RHOA Y42C exhibits impaired GTP hydrolysis and enhances interaction with its effector ROCK. RHOA Y42C mutation and Cdh1 loss induce actin/cytoskeletal rearrangements and activity of focal adhesion kinase (FAK), which activates YAP-TAZ, PI3K-AKT, and β-catenin. RHOA Y42C murine models were sensitive to FAK inhibition and to combined YAP and PI3K pathway blockade. These results, coupled with sensitivity to FAK inhibition in patient-derived DGC cell lines, nominate FAK as a novel target for these cancers. SIGNIFICANCE:The functional signifi cance of recurrent RHOA mutations in DGC has remained unresolved. Through biochemical studies and mouse modeling of the hotspot RHOA Y42C mutation, we establish that these mutations are activating, detail their effects upon cell signaling, and defi ne how RHOA-mediated FAK activation imparts sensitivity to pharmacologic FAK inhibitors.

SUMMARY We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1 , and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.

92 Background: The CCL2-CCR2 signaling axis may facilitate migration of myeloid derived suppressor cells to pancreatic cancer resulting in an immune suppressive tumor microenvironment. CCR2 inhibition in patients with non-metastatic pancreatic cancer was previously reported to decrease tumor-infiltrating macrophages/Treg cells and increase effector T cells (Nywening et al, 2016). Here, a CCR2 specific antagonist CCX872 was used in combination with FOLFIRINOX to treat subjects with locally advanced or metastatic, non-resectable pancreatic cancer in a multi-center study. Methods: Fifty subjects (ECOG score ≤ 2) were enrolled, receiving FOLFIRINOX (fluorouracil [5-FU], leucovorin, irinotecan, oxaliplatin) once every two weeks (maximum 12 cycles) plus 150 mg CCX872 QD or BID for 12 weeks. Subjects showing at least stable disease at the end of the 12-week treatment period were eligible to CCX872 treatment until disease progression. In this ongoing study, all subjects are followed for overall survival (OS). Blood samples were taken at baseline and at intervals throughout the active treatment period for hematologic and flow cytometric analysis of circulating immune cell populations. Results: The all-subjects OS at 18 months is 29%. This compares favorably with previously published data: i.e., OS of only 18.6% at 18 months for FOLFIRINOX regimen alone (Conroy et al, 2011). Peripheral blood monocyte counts at baseline inversely correlate with OS (p = 0.0071, Hazard ratio = 1.169) with CCX872 and FOLFIRINOX combination therapy. Overall, circulating monocytes, inflammatory monocytes and monocytic myeloid derived suppressor cells were reduced by treatment. Conclusions: CCX872-B plus FOLFIRINOX resulted in an OS of 29% at 18 months with no safety issues. Better OS was associated with lower peripheral blood monocyte counts at baseline. Clinical trial information: NCT02345408.

g Cyclin D1-cyclin-dependent kinase 4/6 (CDK4/6) dysregulation is a major contributor to melanomagenesis. Clinical evidence has revealed that p16 INK4A , an allosteric inhibitor of CDK4/6, is inactivated in over half of human melanomas, and numerous animal models have demonstrated that p16 INK4A deletion promotes melanoma. FBXO4, a specificity factor for the E3 ligase that directs timely cyclin D1 proteolysis, has not been studied in melanoma. We demonstrate that Fbxo4 deficiency induces Brafdriven melanoma and that this phenotype depends on cyclin D1 accumulation in mice, underscoring the importance of this ubiquitin ligase in tumor suppression. Furthermore, we have identified a substrate-binding mutation, FBXO4 I377M, that selectively disrupts cyclin D1 degradation while preserving proteolysis of the other known FBXO4 substrate, TRF1. The I377M mutation and Fbxo4 deficiency result in nuclear accumulation of cyclin D1, a key transforming neoplastic event. Collectively, these data provide evidence that FBXO4 dysfunction, as a mechanism for cyclin D1 overexpression, is a contributor to human malignancy. Despite recent advances in immunotherapy and targeted therapy, metastatic melanoma remains an intractable, malignant disease with limited therapeutic options. The most widely appreciated genetic insult associated with melanoma is the constitutive activation of BRAF, a consequence of a valine-to-glutamate substitution at codon 600 (BRAF V600E ) (1-3). The importance of BRAF activation is underscored by its presence in roughly half of all cutaneous melanomas (4) and by the impressive cytotoxicity and tumor regression observed in melanoma patients receiving the BRAF kinase inhibitor vemurafenib (5-7).Attempts to model melanoma initially revealed that BRAF V600E expression in primary melanocytes elicits oncogene-induced senescence (8-10), as opposed to malignant growth. Consistently, ϳ80% of benign human nevi harbor BRAF V600E and never progress to melanoma (4). Thus, while BRAF-driven signaling may be an integral component of melanomagenesis, cooperation with other genetic insults is necessary.Multiple independent lines of evidence indicate that the loss of p16 INK4A , an allosteric inhibitor of cyclin-dependent kinase 4/6 (CDK4/6)-cyclin D, cooperates with RAS-RAF to induce melanoma (11-17). These oncogenic events are clinically significant, as p16INK4A is commonly inactivated in melanomas (18,19). In addition to p16 INK4A , the cyclin D1 gene (CCND1) is amplified in multiple melanoma subtypes (over 40% of acral melanomas) (20), also contributing to dysregulated CDK4/cyclin D1 activity.While amplification events contribute to increased cyclin D1 expression, roughly 20% of melanomas overexpressing cyclin D1 do not exhibit genetic alterations in CCND1 (20). The latter observation suggests that dysregulated posttranslational control may contribute to cyclin D1 overexpression. However, the role of the degradation machinery for cyclin D1, a highly labile protein (21), has not been extensively studied outside the contex...

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Highlights d Pancreatic cancer cells are dependent on CHEK1 and DNA damage response genes d CHK1 inhibition causes compensatory ERK and autophagy activation d Concurrent CHK1 and ERK inhibition causes synergistic loss of MYC