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Supplementary Figure 1 D-H from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Supplementary Figure 4 from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Supplementary Figure 3 from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Supplementary Figures 1-4, Table 1 from Multiple Roles for the Receptor Tyrosine Kinase Axl in Tumor Formation

Supplementary Figure 6 from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Supplementary Figure 1 A-C from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Supplementary Tables 1-3 from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Supplementary Figure and Table Legends from Multiple Roles for the Receptor Tyrosine Kinase Axl in Tumor Formation

Supplementary Figure 5 from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Supplementary Figure 2 from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Supplementary Materials, Figure Legends 1-6 from R428, a Selective Small Molecule Inhibitor of Axl Kinase, Blocks Tumor Spread and Prolongs Survival in Models of Metastatic Breast Cancer

Introduction: In normal tissue homeostasis, interaction of phosphatidylserine externalized on apoptotic cells (ACs) with the TAM (Tyro3, Axl MerTK) family RTK MerTK, via its ligands Gas6 and Protein S, leads to AC phagocytosis (efferocytosis). The resulting clearance of AC antigens, immunosuppressive M2 macrophage polarization, and suppression of pro-inflammatory cytokine production promotes tolerance to AC-derived self-antigens. This homeostatic response is coopted in tumors, which are abundant in both ACs and TAM ligands, leading to a blunted anti-tumor immune response. Syngeneic tumors implanted in MerTK -/- mice exhibit poor growth and metastasis, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10 compared with those implanted in WT mice. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens and promoting an anti-tumor immune response, or by directly blocking tumor cell survival. We have therefore developed small molecule inhibitors of MerTK. Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell number, apoptosis and proliferation. Effects on immune function were tested in human primary dendritic cells (LPS-induced IL-23 production) and human primary T cells (anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5). Efferocytosis of apoptotic Jurkat cells by human primary macrophages was detected by flow cytometry. For the PD assay, MerTK expressing tumors were grown in nude mice. 30-60 minutes post-compound dosing, MerTK was stimulated in vivo for 1hr. Tumors were snap frozen and tumor lysates were blotted with anti-phospho-MerTK antibodies. Anti-tumor efficacy was studied in syngeneic models. Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both classes of compounds with EC50 Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response. Note: This abstract was not presented at the conference. Citation Format: Sacha J. Holland, Alexander M. Owyang, Sylvia Braselmann, Chrystelle Lamagna, Sothy Yi, Chi Young, Roy Frances, Arthur Bagos, Meagan Chan, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Jiaxin Yu, Ihab Darwish, Somasekhar Bhamidipati, Donald G. Payan, Esteban Masuda. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B060.

Recent advances in genome sequencing and tumor proteome and transcriptome analysis uncovered a key role for MyD88-dependent Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R)-mediated signaling pathways in multiple hematologic malignancies. A third of activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) and nearly 100% of Waldenstrom macroglobulinemia (WD) patients carry an activating Myd88 L265P mutation. Overexpression of multiple TLR pathway components, associated with deregulation of innate immune system and subsequent induction of proinflammatory bone marrow environment, are prominent features of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). IRAK4 kinase is a crucial enzyme in all MyD88-dependent signaling pathways, potentially making it an ideal target for disease modification with small molecule inhibitors. Through cell-based screening, we identified a potent small molecule IRAK1/4 kinase inhibitor, R191. R191 blocks TLR- and IL-1R-induced cytokine production in primary cells with potencies below 50nM while sparing unrelated pathways with at least 20-fold window. R191 is extremely potent in vitro against IRAK4 kinase (3 nM), yet exhibits good selectivity against a broad panel of kinases. In vivo, it decreases serum IL-6 in an acute mouse model of IL-1β-induced cytokine release and blocks joint inflammation in the collagen-induce arthritis model. R191 exhibited strong synergy with Bcl2 and BCR pathway inhibitors against a number of DLBCL lines in vitro. In multiple AML lines, R191 potently inhibited expression of PD-L1, potentially promoting recognition of AML blasts by the immune system. Based on the potential critical role of IRAK kinases in MDS, AML and Myd88 L265P lymphomas, R191 could provide a novel therapeutic modality in the management of multiple inflammation-driven hematologic malignancies. Citation Format: Vadim V. Markovtsov, Chrystelle Lamagna, Meagan Chan, Sothy Yi, Chi Young, Roy Frances, Stacey Siu, Sylvia Braselmann, Hui Li, Rajinder Singh, Gary Park, Esteban Masuda, Vanessa Taylor, Donald G. Payan. Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 346.

5’-AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and is critical for maintaining energy homeostasis under conditions of nutrient stress. During cellular transformation, metabolic reprogramming enables the aberrant growth and proliferation of tumor cells. Both positive and negative roles for AMPK in tumor cell proliferation and survival have been reported. However, only a limited number of studies addressed this question with potent direct AMPK activators. AMPK exists as heterotrimers composed of the catalytic subunit α and reguratory subunits β and γ. We expressed the full length of all three human AMPK subunits in insect cells, purified the heterotrimer complexes, and used them for biochemical screening and characterization of AMPK activators. The purified complexes displayed basal activity, which was further enhanced by AMP. The compounds we identified potently activated the complexes in vitro at AC(2X)s (the concentration that gives a twofold activation) of 0.001-0.3 μM. Importantly, the compounds up-regulated substrate phosphorylation (pS79 Acetyl-CoA Carboxylase) and/or auto-phosphorylation (pT172 AMPKα) in multiple cancer cell lines including HepG2 hepatoma cells, A549 liver kinase B1 (LKB1) null lung cancer cells, and MOLM14 myeloid leukemia cells, indicating activation was irrespective of functional status of LKB1, which is a key AMPK-activation kinase. Activation of AMPK by the compounds was also confirmed using native AMPK isolated from normal tissues and tumor cells. We further investigated anti-proliferative effects of the compounds and found that up-regulation of AMPK kinase activity was correlated with anti-proliferative effects in A549 and MOLM14, but not in HepG2, suggesting that positive effects of direct AMPK activators could be cell-type dependent. Interestingly, we identified compounds that display comparable AMPK activation in HepG2 and A549 yet possessed divergent activities on proliferation across a panel of tumor lines. Analysis of cellular signaling across several of these tumor lines with this set of the compounds revealed dose-dependent effects on mTORC1 substrates, feedback signaling to PI3K and mTORC2, and inhibition of kinases downstream of RAF. Direct activation of AMPK could be a good therapeutic strategy for the treatment of subsets of cancers. Citation Format: Yasumichi Hitoshi, Yonchu Jenkins, Yingwu Li, Elmer Sampang, Xiang Xu, Guodong Dong, Jianing Huang, Nan Lin, Dane Goff, Simon Shaw, Luke Boralsky, Rajinder Singh, Sarkiz D. Issakani, Donald G. Payan. Development of small molecule direct AMPK activators for the treatment of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3021.

Introduction: MerTK, a TAM (Tyro3, Axl, MerTK) family RTK, is expressed on phagocytic myeloid and epithelial cells. Its normal function is to dampen innate immune responses to self-antigens. MerTK is an indirect phosphatidylserine (PtdSer) receptor: PtdSer-binding TAM ligands, Gas6 or Protein S, bridge interactions between MerTK and PtdSer externalized on apoptotic cells (ACs), resulting in AC internalization (efferocytosis). Ensuing MerTK signaling leads to anti-inflammatory M2 macrophage polarization, suppression of pro-inflammatory cytokine production, and a tolerogenic outcome. Tumors are rich in ACs and TAM ligands. Syngeneic tumors implanted in MerTK -/- mice exhibit impaired growth and metastasis compared with those implanted in WT mice, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens, blocking tumor induced immunosuppression, or directly promoting tumor cell survival. We have therefore developed small molecule inhibitors of MerTK. Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell proliferation, DNA content and apoptosis. Immune effector assays were LPS-induced IL-23 production in human primary dendritic cells and anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5 in human primary T cells. Efferocytosis of CFSE-labeled apoptotic Jurkat cells by anti-CD14-labeled human primary macrophages was detected by flow cytometry. Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both compounds with EC50 Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response. Citation Format: Sacha J. Holland, Alex M. Owyang, Sothy Yi, Chi Young, Sylvia Braselmann, Roy Frances, Arthur Bagos, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Somasekhar Bhamidipati, Ihab Darwish, Matthew Duncton, Rajinder Singh, Esteban Masuda, Donald G. Payan. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4869.

Background: Metastasis and drug resistance are the major causes of mortality in patients with pancreatic ductal adenocarcinoma (PDA). Once developed, the progression of PDA metastasis is virtually unstoppable with current therapies. Accumulating evidence suggests important roles for the receptor tyrosine kinase AXL in cancer progression, invasion, metastasis, drug resistance, and patient mortality. Given the critical influence of metastasis on the clinical history of PDA, we hypothesize that pharmacological inhibition of AXL might inhibit tumor growth and prevent PDA metastasis. Methods: We investigated the AXL expression in a panel of human pancreatic cancer xenografts using Affymetrix gene array as well as qRT-PCR. We conducted a comprehensive preclinical trial to determine whether AXL targeted monotherapy, and in combination with gemcitabine, was effective in blocking PDA tumor progression and metastasis using subcutaneous as well as orthotopic pancreatic cancer models. Results: R428, a potent selective small molecule inhibitor of AXL, inhibited proliferation of pancreatic cancer cell lines in a concentration-dependent manner. A combination of R428 with gemcitabine could significantly inhibit primary tumor growth compared to gemcitabine treated mice. Notably, R428 alone and in combination with gemcitabine was remarkably effective in preventing the development of metastasis to organs and lymph nodes as compared to gemcitabine treatment in a highly aggressive orthotopic model. Conclusions: The data presented here provide strong evidence for the therapeutic development of AXL inhibitors as anti-metastatic agents in pancreatic cancer. A combination treatment of R428 and gemcitabine was highly effective in controlling the primary tumor growth and preventing metastatic spread. The novel therapeutic approach tested here has potential therapeutic implications for improving the current standard of care for PDA patients and to contribute towards making PDA a more manageable disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2546. doi:10.1158/1538-7445.AM2011-2546