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Supplementary Figure S1 shows genetic dependency profiles for BRAF, MAPK1 (ERK2), and MAPK3 (ERK1) by BRAFV600 mutation status from three independent screens: DRIVE, ACHILLES, and AVANA.

All supplementary figure and table titles and legends.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues.Implications:BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing.

Purpose: PIM kinases have been shown to act as oncogenes in mice, with each family member being able to drive progression of hematologic cancers. Consistent with this, we found that PIMs are highly expressed in human hematologic cancers and show that each isoform has a distinct expression pattern among disease subtypes. This suggests that inhibitors of all three PIMs would be effective in treating multiple hematologic malignancies.Experimental Design: Pan-PIM inhibitors have proven difficult to develop because PIM2 has a low Km for ATP and, thus, requires a very potent inhibitor to effectively block the kinase activity at the ATP levels in cells. We developed a potent and specific pan-PIM inhibitor, LGB321, which is active on PIM2 in the cellular context.Results: LGB321 is active on PIM2-dependent multiple myeloma cell lines, where it inhibits proliferation, mTOR-C1 signaling and phosphorylation of BAD. Broad cancer cell line profiling of LGB321 demonstrates limited activity in cell lines derived from solid tumors. In contrast, significant activity in cell lines derived from diverse hematological lineages was observed, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma and non-Hodgkin lymphoma (NHL). Furthermore, we demonstrate LGB321 activity in the KG-1 AML xenograft model, in which modulation of pharmacodynamics markers is predictive of efficacy. Finally, we demonstrate that LGB321 synergizes with cytarabine in this model.Conclusions: We have developed a potent and selective pan-PIM inhibitor with single-agent antiproliferative activity and show that it synergizes with cytarabine in an AML xenograft model. Our results strongly support the development of Pan-PIM inhibitors to treat hematologic malignancies. Clin Cancer Res; 20(7); 1834–45. ©2014 AACR.

PDF file - 346KB, Supplementary Figure 1. Normal vs. Tumor expression of PIM kinases mRNA across 17 tissue types. Supplementary Figure 2. Comparison of LGB321 on-target activity vs. Previously described pan-PIM inhibitor. Supplementary Figure 3. KINOMESCANTM of 1 mu M LGB321. Supplementary Figure 4. Biochemical and cellular selectivity of the LGB321 PIM inhibitor series towards GSK3 beta. Supplementary Figure 5. LGB321 does not inhibit EGFR signaling in HCC1954 cells. Supplementary Table 1. Sensitivity to LGB321 and Erlotinib of Lung Cells in the CCLE. Supplementary Table 2. Sensitivity to LGB321 of hematological malignacies cell lines.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues. Implications: BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing.

Overexpression of PIM 1, 2, and 3 kinases is frequently observed in many malignancies. Previously, we discovered a potent and selective pan-PIM kinase inhibitor, compound 2, currently in phase I clinical trials. In this work, we were interested in replacing the amino group on the cyclohexane ring in compound 2 with a hydroxyl group. Structure-based drug design led to cellularly potent but metabolically unstable tetra-substituted cyclohexyl diols. Efforts on the reduction of Log D by introducing polar heterocycles improved metabolic stability. Incorporating fluorine to the tetra-substituted cyclohexyl diol moiety further reduced Log D, resulting in compound 14, a cellularly potent tetra-substituted cyclohexyl diol inhibitor with moderate metabolic stability and good permeability. We also describe the development of efficient and scalable synthetic routes toward synthetically challenging tetra-substituted cyclohexyl diol compounds. In particular, intermediate 36 was identified as a versatile intermediate, enabling a large-scale synthesis of highly substituted cyclohexane derivatives.

Structure-based modification of mifepristone (1) led to the discovery of novel mifepristone derivatives with improved selectivity profile. Addition of a methyl group at the C10 position of the steroid has a significant impact on progesterone receptor (PR) and androgen receptor (AR) activity. Within this series, OP-3633 (15) emerged as a glucocorticoid receptor (GR) antagonist with increased selectivity against PR and AR, improved cytochrome P450 inhibition profile, and significantly improved pharmacokinetic properties compared to 1. Furthermore, 15 demonstrated substantial inhibition of GR transcriptional activity in the GR positive HCC1806 triple negative breast cancer xenograft model. Overall, compound 15 is a promising GR antagonist candidate to clinically evaluate the impact of GR inhibition in reversal or prevention of therapy resistance.

Adenosine accumulation within the tumor microenvironment can severely limit antitumor immunity by promoting the expansion of immunosuppressive cell types and impairing immune cell function, including T cells, natural killer cells and dendritic cells. The generation of adenosine from adenosine monophosphate (AMP) requires the activity of a cell surface ecto-5’-nucleotidase, CD73, expressed on the surface of various cell types including immune cells. CD73 overexpression is observed in many tumor types and correlates with unfavorable clinical outcomes. Given the essential role of CD73 in generating adenosine, inhibition of CD73 is a promising therapeutic strategy. Recent clinical proof of concept data demonstrated a significant improvement in progression free survival for non-small cell lung cancer patients upon targeting CD73 with the oleclumab anti-CD73 antibody in combination with anti-PDL1, relative to anti-PDL1 checkpoint inhibitor treatment alone.We developed ORIC-533, a potent, orally bioavailable, AMP-competitive, small molecule inhibitor of CD73, that is highly selective for CD73 and exhibits picomolar potency in biochemical assays, completely blocking adenosine production from AMP. In ex vivo assays, ORIC-533 displayed greater potency in restoring immunosuppressed CD8+ T cell proliferation and activation compared to oleclumab, as well as greater potency versus small molecule inhibitors of CD73 and the adenosine receptor. In multiple myeloma (MM) patients, elevated adenosine levels in the bone marrow (BM) niche correlate with progression of multiple myeloma through a CD73-mediated pathway. Moreover, we recently reported a functional role of CD73 signaling in BM of MM patients, indicating that CD73 inhibition may represent a unique vulnerability and treatment strategy for MM (Ray et al., abstract #2675, ASH 2021). We tested the impact of CD73 inhibition in bone marrow aspirates from patients with relapsed or refractory MM, using an autologous ex vivo assay system comprised of the multiple myeloma BM microenvironment. CD73 inhibition stimulated the activation of plasmacytoid dendritic cells and T cell activation in the context of the MM BM milieu. Moreover, ORIC CD73 inhibitor as a single agent overcame immune suppression and triggered significant lysis and cell death of multiple myeloma cells by autologous T-cells in the bone marrow microenvironment. Taken together, these results demonstrate that the ORIC CD73 inhibitor potently inhibits the adenosine pathway, which restores anti-tumor immunity and therefore holds potential for patients with MM. Based upon these data, ORIC-533 is being studied as a single agent in a Phase 1 clinical trial in patients with relapsed or refractory multiple myeloma. Citation Format: Melissa R. Junttila, Arghya Ray, Robert Warne, Xi Chen, Ting Du, Dena Sutimantanapi, Jae H. Chang, Brian Blank, Jared Moore, Chudi O. Ndubaku, Tatiana Zavorotinskaya, Pratik Multani, Omar Nadeem, Dharminder Chauhan, Kenneth C. Anderson, Lori S. Friedman. ORIC-533, a small molecule CD73 inhibitor with best-in-class properties, reversesimmunosuppression and has potential as an immunomodulatory therapy in patients with multiple myeloma [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 2074.

The adenosinergic pathway represents an attractive new therapeutic approach in cancer immunotherapy. In this pathway, ecto-5-nucleotidase CD73 has the unique function of regulating production of immunosuppressive adenosine (ADO) through the hydrolysis of AMP. CD73 is overexpressed in many cancers, resulting in elevated levels of ADO that correspond to poor patient prognosis. Therefore, reducing the level of ADO via inhibition of CD73 is a potential strategy for treating cancers. Based on the binding mode of adenosine 5'-(α,β-methylene)diphosphate (AOPCP) with human CD73, we designed a series of novel monophosphonate small-molecule CD73 inhibitors. Among them, OP-5244 (35) proved to be a highly potent and orally bioavailable CD73 inhibitor. In preclinical studies, 35 completely inhibited ADO production in both human cancer cells and CD8+ T cells. Furthermore, 35 lowered the ratio of ADO/AMP significantly and reversed immunosuppression in mouse models, indicating its potential as an in vivo tool compound for further development.

The glucocorticoid receptor (GR) has been linked to therapy resistance across a wide range of cancer types. Preclinical data suggest that antagonists of this nuclear receptor may enhance the activity of anticancer therapy. The first-generation GR antagonist mifepristone is currently undergoing clinical evaluation in various oncology settings. Structure-based modification of mifepristone led to the discovery of ORIC-101 (28), a highly potent steroidal GR antagonist with reduced androgen receptor (AR) agonistic activity amenable for dosing in androgen receptor positive tumors and with improved CYP2C8 and CYP2C9 inhibition profile to minimize drug-drug interaction potential. Unlike mifepristone, 28 could be codosed with chemotherapeutic agents readily metabolized by CYP2C8 such as paclitaxel. Furthermore, 28 demonstrated in vivo antitumor activity by enhancing response to chemotherapy in the GR+ OVCAR5 ovarian cancer xenograft model. Clinical evaluation of safety and therapeutic potential of 28 is underway.

Amplification of human epidermal growth factor receptor 2 (HER2) is an oncogenic driver found in approximately 25% of breast cancer. Despite the arsenal of HER2-directed therapies available to patients, more than 50% of patients with HER2 amplification eventually develop central nervous system (CNS) metastases over the course of their disease indicating a clear medical need for brain penetrant therapies in this patient population. ORIC-114 is a brain penetrant, orally bioavailable, irreversible small molecule inhibitor that was designed to target exon20 insertions in epidermal growth factor receptor (EGFR) and HER2. ORIC-114 is highly selective for the EGFR/HER2 family of receptors, reducing the risk for off-target kinase liabilities. In biochemical assays, ORIC-114 displayed low nanomolar potency on HER2. To explore the application of ORIC-114 in the HER2-amplified tumor setting, a cell viability screen was performed against a panel of human breast cancer lines containing both HER2-amplified and non HER2-amplified cell lines. ORIC-114 demonstrated greater than 100-fold enhanced cellular potency on HER2-amplified cancer cell lines relative to non-amplified cancer cell lines. Notably, ORIC-114 cellular EC50s in HER2-amplified breast cancer cell lines were below 30 nM and more potent than both lapatinib and tucatinib, two FDA-approved tyrosine kinase inhibitors for the treatment of HER2-positive breast cancer. ORIC-114 was designed to incorporate physicochemical properties suitable to cross the blood-brain barrier and has exhibited good brain penetration across multiple preclinical species. To further investigate the brain penetrant attributes of ORIC-114 in the context of HER2-positive breast cancer with brain metastases, key features were assessed relative to tucatinib, which has demonstrated clinical efficacy in this setting. In contrast to tucatinib, ORIC-114 displayed minimal impact on efflux transporters as it was only a weak substrate for P-glycoprotein (P-gp) and was not a substrate for breast cancer associated protein (BCRP) in vitro. In addition, ORIC-114 demonstrated superior in vivo brain penetration relative to tucatinib as measured by free brain-to-plasma ratio in mouse. Consequently, ORIC-114 free brain concentrations in rodents were greater than tucatinib, even when the active metabolites of tucatinib were considered. Taken together, these data further establish ORIC-114 as a selective, irreversible, and brain penetrant EGFR/HER2 inhibitor, making it a promising therapeutic candidate for development in patients with HER2-positive tumors including those with CNS metastases. A Clinical Trial Application for ORIC-114 is anticipated in the second half of 2021. Citation Format: Melissa R. Junttila, Jason E. Long, Robert Warne, Sunghwan Kim, Younho Lee, Hwan Kim, Juhee Kang, Jiyoon Seok, Jihye Yoo, Youngyi Lee, Dong-Hyuk Seo Seo, Jung Beom Son, Daekwon Kim, Hwan Geun Choi, Nam Doo Kim, Tatiana Zavorotinskaya, Chelsea Chan, Matthew Panuwat, Jessica Sun, Jae H. Chang, Lori S. Friedman. ORIC-114, an orally bioavailable, irreversible kinase inhibitor, has superior brain penetrant properties and enhanced potency in preclinical studies of HER2-positive breast cancer [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 P234.

Introduction: Adenosine is an anti-inflammatory and immunosuppressive metabolite, that signals to diminish activation and proliferation of cytotoxic T-cells, impair activity of natural killer cells and CD4 + effector T-cells, and promote the expansion of immunosuppressive cell types. CD73, a cell surface ecto-5'-nucleotidase, is required to convert AMP to adenosine and is a major catalyst of adenosine generation in the tumor microenvironment. Overexpression of CD73 is observed in many tumors and correlates with unfavorable clinical outcome. Bone marrow (BM) aspirates from multiple myeloma (MM) patients have shown increased adenosine levels correspond with disease progression [Horenstein et al. Mol Med. 2016,22:694-704] In addition to the adenosine rich feature of MM, multiple cell types within the MM BM niche express the enzymes required for adenosine production from both NAD and ATP precursors, including CD38, CD203a, CD39 and CD73. Previously, we demonstrated that dysfunctional plasmacytoid dendritic cells (pDCs) predominantly found in the BM of MM patients contribute to MM cell growth, survival, and suppression of antitumor immunity [Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia 2015, 29:1441-1444]. We recently discovered that the interaction between pDCs and MM cells increased CD73 transcript and protein levels in both cell types, implicating a role for adenosine signaling via CD73 signaling axis in MM. Together, these MM disease features indicate that reducing the level of adenosine via inhibition of CD73 may represent a unique vulnerability and treatment strategy for MM. Methods: To understand the functional consequence of CD73 inhibition in MM, autologous ex vivo cell assays using freshly isolated BM aspirates from MM patients were used to detect changes in immune cell function and MM cell viability upon treatment with OP-5558, a potent and selective CD73 small molecule inhibitor which is an analog of the clinical candidate, ORIC-533. The majority of BM samples utilized were from patients with relapsed or refractory MM after at least three lines of therapy including immunomodulatory drugs, proteasome inhibitors, and anti-CD38 monoclonal antibodies, as well as a patient with relapsed MM post BCMA-CAR-T therapy. Results: In BM aspirates from MM patients with relapsed refractory MM, CD73 inhibition by OP-5558 triggered activation of MM pDCs, evidenced by increased expression of CD40/CD83/CD86 (1.2-1.5-fold, OP-5558-treated versus untreated; p < 0.05; n=3). This inhibition of CD73 reversed immunosuppression in MM BM. Specifically, CD73 inhibitor OP-5558 stimulated T-cell activation, associated with increased CD69 cell surface expression on CD3 + T-cells (CD69 MFI:20% increase, treated versus control; p = 0.0031; n = 3). Moreover, CD8 + T-cells from these co-cultures enhanced cytolytic activity against patient MM cells, significantly decreasing autologous MM cell viability (mean 42% decrease in viability; treated versus control; p=0.014; n=5). Of note, OP-5558 treatment did not directly affect viability of MM cells when treated in isolation, indicating that the observed decreased viability occurs via enhanced cytotoxic T-cell activity. Importantly, we show that OP-5558 triggered significant MM cell lysis even within autologous MM bone marrow mononuclear cell (BMNC) cultures, confirming that CD73 inhibition restores MM-specific cytolytic activity of autologous patient T-cells in the MM BM microenvironment. (mean 37% decrease in viability; treated versus control; p=0.009; n=3). Conclusions: This study therefore demonstrates that: 1. CD73-mediated adenosine activity suppresses the cytolytic activity of T-cells against tumor cells in the MM BM milieu; and 2. CD73 inhibition can overcome immune suppression and restore lysis of MM cells by autologous T-cells. A clinical trial of potent, selective, orally bioavailable CD73 inhibitor ORIC-533 will examine the utility of CD73 inhibition to improve outcome in patients with relapsed refractory MM. Disclosures Junttila: ORIC Pharmaceuticals: Current Employment. Sutimantanapi: ORIC Pharmaceuticals: Current Employment. Chen: ORIC Pharmaceuticals: Current Employment. Warne: ORIC Pharmaceuticals: Current Employment. Chang: ORIC Pharmaceuticals: Current Employment. Blank: ORIC Pharmaceuticals: Current Employment. Wu: ORIC Pharmaceuticals: Current Employment. Moore: ORIC Pharmaceuticals: Current Employment. Ndubaku: ORIC Pharmaceuticals: Current Employment. Zavorotinskaya: ORIC Pharmaceuticals: Current Employment. Nadeem: GSK: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Friedman: ORIC Pharmaceuticals: Current Employment. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Adenosine is recognized for its anti-inflammatory and immunosuppressive properties. Signaling through adenosine receptors expressed on immune cells initiates a cascade that diminishes activation and proliferation of cytotoxic T-cells, impairs activity of natural killer cells and CD4+ effector T-cells, and promotes the expansion of immunosuppressive cell types. CD73, a cell surface ecto-5'-nucleotidase, converts AMP to adenosine and is a major catalyst of adenosine generation in the tumor microenvironment. Overexpression of CD73 is observed in many solid tumors and may correlate with unfavorable clinical outcomes; therefore, reducing the level of adenosine via inhibition of CD73 is a strategy for enhancing antitumor immune responses and overcoming therapeutic resistance. We have developed ORIC-533, a potent, orally bioavailable, AMP-competitive, small molecule inhibitor of CD73. ORIC-533 is highly selective for CD73 and exhibits picomolar potency in biochemical and cellular assays, blocking adenosine production from AMP. Expression of CD73 on immune cells is sufficient to drive immunosuppression in vitro when AMP is present. We have previously shown that ORIC-533 can rescue activation of CD8+ T- cells exposed to AMP and restore proliferation, cytokine secretion and CD25 expression at low nanomolar concentrations. To understand the role of CD73 in dendritic cell (DC) priming of helper CD4+ T-cells, we utilized co-culture of allogeneic immature DC and CD4+ T-cells, where AMP to adenosine conversion severely suppresses activation of helper CD4+ T-cells. Treatment with ORIC-533 completely restored activation of CD4+ T-cells in this setting. To better define the biologically relevant AMP concentrations likely to be encountered in tumors, we determined the concentration of AMP in a panel of primary patient tumors. We discovered that intratumoral AMP levels can reach up to 500 micromoles, emphasizing the requirement for CD73 inhibitors to be efficacious in a high AMP environment. We previously demonstrated that nanomolar concentrations of ORIC-533, unlike other CD73 inhibitors tested, can efficiently rescue cytotoxic T-cell function in the presence of millimolar AMP concentrations. Surprisingly, in these studies we found that inhibitors of adenosine receptors, A2A or A2A/B, can only rescue CD8+ T-cell function in the context of low micromolar AMP or adenosine and thus are likely to be ineffective in patient tumors with high AMP. Taken together, these preclinical results indicate the CD73 inhibitor ORIC-533 has best-in-class properties in reversing immunosuppression in tumors. ORIC-533 is currently undergoing GLP studies to support an IND filing in the first half of 2021. Citation Format: Dena Sutimantanapi, Chelsea Chen, Bob Warne, Alex Pankov, Jae Chang, Lori Friedman, Melissa Junttila, Tatiana Zavorotinskaya. Blocking adenosine production with ORIC-533, a CD73 inhibitor with best-in-class properties, reverses immunosuppression in high-AMP environments [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 LB163.

Genomic insertions within exon20 of both epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are oncogenic drivers most commonly found in non-small cell lung cancer (NSCLC) but also occurring in multiple other tumor types. Exon20 insertions render the receptors resistant to currently approved inhibitors, giving patients with tumors harboring such insertions a worse prognosis than with other activating EGFR mutations. Moreover, approximately one-third of patients with exon20 insertion mutations may develop central nervous system (CNS) metastases over the course of their disease. To address this unmet medical need, ORIC-114, a brain penetrant, orally bioavailable, irreversible small molecule inhibitor was designed to target exon20 insertions in EGFR and HER2. ORIC-114 is highly selective to the EGFR family of receptors, showing superior kinome selectivity compared with other reported exon20 inhibitors. In biochemical assays ORIC-114 displays low nanomolar potency, and importantly has enhanced potency on the EGFR exon20 NPG insertion relative to wildtype EGFR protein. ORIC-114 also demonstrates low nanomolar potency across exon20 insertion variants using cell-based assays measuring EGFR phosphorylation, downstream signaling and cell viability. ORIC-114 is readily brain available across multiple preclinical species hence studies were undertaken to investigate activity in both subcutaneous and intracranial tumor models. Herein, we explored the in vivo activity of ORIC-114. Consistent with our in vitro findings, robust activity was observed in EGFR exon20 patient-derived xenograft models using once daily oral administration, with greater than 90% tumor growth inhibition in the absence of body weight loss. Moreover, these significant antitumor effects correlate with decreased pharmacodynamic response as measured by phosphorylated EGFR in terminal tumors. To investigate whether the brain-penetrant attributes of ORIC-114 translate into therapeutic CNS activity, we utilized an intracranial luciferase-labeled EGFR mutant cell line model. Once daily oral administration of ORIC-114 significantly regressed established intracranial NSCLC tumors, demonstrating greater efficacy than TAK-788, commensurate with the superior brain to plasma exposure of ORIC-114. Taken together, these data establish ORIC-114 as a selective, irreversible, and brain penetrant EGFR inhibitor, making it a promising therapeutic candidate for development in patients with tumors harboring EGFR and HER2 exon20 insertions, including those with CNS metastases. ORIC-114 is anticipated to enter a global Phase 1/2 tumor-agnostic trial in genetically defined cancers in the second half of 2021. Citation Format: Melissa R. Junttila, Sunghwan Kim, Younho Lee, Hwan Kim, Juhee Kang, Jiyoon Seok, Jihye Yoo, Youngyi Lee, Dong-Hyuk Seo, Jung Beom Son, Daekwon Kim, Hwan Geun Choi, Nam Doo Kim, Akash Das, Dena Sutimantanapi, Tatiana Zavorotinskaya, Chelsea Chen, Jae Chang, Matthew Panuwat, Lori Friedman. ORIC-114, a brain penetrant, orally bioavailable, irreversible inhibitor selectively targets EGFR and HER2 exon20 insertion mutants and regresses intracranial NSCLC xenograft tumors [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 1466.

The 5' ecto-nucleotidase CD73 is dynamically expressed in distinct biological contexts as a means to increase the adenosine metabolite, which functions to limit immune activation and prevent excessive inflammation. CD73 activity is required for the final step of adenosine production. In the context of tumors, excessive CD73-mediated adenosine generation impairs anti-tumor immune responses. Here, we show that an orally bioavailable small molecule inhibitor of CD73 can prevent adenosine generation, relieve adenosine-driven immunosuppression within the tumor in vivo and generate anti-tumor efficacy. In vitro analysis confirmed the substantial suppressive effects of adenosine on murine CD8+ T cells, cross-presenting dendritic cells, and macrophages. NECA, a stable analog of adenosine, impaired immature bone-marrow precursor differentiation into CD103+ dendritic cells, a subset which is critical for tumor-derived antigen presentation to naïve T cells. Interestingly, NECA also inhibited the ability of macrophages to upregulate the M1 marker, CD80, in both the presence and absence of additional polarizing stimuli. Furthermore, OR-558, a CD73 small molecule inhibitor, could fully inhibit CD73-mediated adenosine production and completely restore T cell activation at sub-nanomolar concentrations. Prior work in CD73 germline knock-out animals has shown that EG7 syngeneic tumor growth is highly dependent on CD73. Building on this knowledge, we next we sought to evaluate CD73 inhibition on AMP to adenosine conversion ex vivo in EG7 syngeneic tumor extracts and indeed observed that OR-558 treatment could significantly impair adenosine production within the tumor milieu. This finding translated to the in vivo tumor setting, where single agent anti-tumor efficacy was observed with continuous OR-558 treatment. Anti-tumor efficacy coincided with significantly decreased intra-tumoral adenosine levels and immune modulation, such as increased intratumoral T cell activation, dendritic cell maturation, and M1 macrophage polarization. Lastly, we determined the bioavailability of orally dosed OR-558 in EG7 tumor-bearing mice and found that significant single agent anti-tumor efficacy was also achievable with once-daily oral dosing of OR-558. Taken together, these data demonstrate that adenosine has pleiotropic immunosuppressive effects and inhibition of CD73-mediated adenosine production in vivo is sufficient to reverse these immune effects and enhance anti-tumor immunity. These results support clinical development of orally bioavailable CD73 small molecule inhibitors. Citation Format: Todd Metzger, Brian Blank, Brenda Chan, Chelsea Chen, Yuping Chen, Xiaohui Du, Frank Duong, Lori Friedman, Melissa Junttila, Hiro Kawai, Wayne Kong, Jared Moore, Johnny Pham, Yosup Rew, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Kejia Wu, Chien-Hung Yeh, Natalie Yuen, Tatiana Zavorotinskaya. An orally bioavailable inhibitor of CD73 reverts intratumoral immunosuppression and promotes anti-tumor responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-115.

CD73 has emerged as an attractive target for cancer immunotherapy. CD73 (ecto-5-nucleotidase) is a glycosyl phosphatidyl inositol (GPI)-anchored cell surface protein and catalyzes the hydrolysis of AMP into immunosuppressive adenosine and inorganic phosphate. CD73 is widely overexpressed in tumor microenvironments (TME) of many cancers, resulting in elevated levels of extra cellular adenosine (ADO). ADO plays a critical role in tumor progression through immune suppression, chemotherapy resistance, metastasis and angiogenesis. Therefore, reducing the level of adenosine via inhibition of CD73 has become a potential strategy for treating cancers. Here we report our medicinal chemistry efforts in developing orally available small molecule CD73 inhibitors. Based on the binding mode of adenosine 5'-(α,β-methylene)diphosphate (APCP) with CD73, we designed a novel series of monophosphate CD73 inhibitors, which are highly potent and orally bioavailable. In preclinical studies, OP-5244 inhibited ADO production completely in human cancer cells and CD8+ T cells. It also showed dose-dependent inhibitory effects on CD73 activity in various tumors ex vivo. Furthermore, OP-5244 showed PK/PD efficacy through lowering of ADO/AMP ratio and reversal of immunosuppression in vivo. Citation Format: Xiaohui Du, Brian Blank, Brenda Chan, Xi Chen, Yuping Chen, Frank Duong, Lori Friedman, Tom Huang, Melissa R. Junttila, Wayne Kong, Todd Metzger, Jared Moore, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Natalie Yuen, Tatiana Zavorotinskaya. Orally available small molecule CD73 inhibitor reverses immunosuppression through blocking of adenosine production [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1037.

Adenosine has emerged as a key immunosuppressive metabolite within the tumor microenvironment (TME). A persistently elevated concentration of adenosine in TME can impair immune control of tumor growth by diminishing cytotoxic T-cell responses and function of natural killer cells while augmenting the suppressive activity of myeloid and regulatory T-cells. The ectonucleotidase, CD73 mediates the final dephosphorylation step in the conversion of extracellular ATP to adenosine. Overexpression of CD73 is observed in many solid tumors and correlates with unfavorable clinical outcome. We aimed to overcome adenosine-driven immunosuppression by developing an orally bioavailable, best-in-class, small molecule inhibitor of CD73. Here, we show superior potency of our selective AMP-competitive CD73 inhibitor in blocking adenosine generation by multiple cell types. CD73 inhibitor activity was assessed by directly measuring the generation of adenosine from AMP by LC-MS/MS. In CD73 expressing cells, potent and complete inhibition of CD73 ectonucleotidase activity was observed. To test the functional consequence of CD73 inhibition, we interrogated the effects of CD73 inhibitors on CD8+ T-cell function upon exposure to AMP. CD73 expressed on T-cells is sufficient to drive adenosine generation from AMP resulting in severely suppressed proliferation, diminished production of inflammatory cytokines and reduced expression of activation markers. Our inhibitors successfully counteracted the effects of AMP and completely rescued all aspects of CD8+ T-cell activation. Interestingly, we found that the concentration of AMP in tumors may vary from micromolar to millimolar levels, underscoring the necessity of AMP-competitive CD73 inhibitors to be efficacious in a high AMP environment. We demonstrated that nanomolar concentrations of our CD73 inhibitors can efficiently rescue T-cell function in the presence of millimolar AMP concentrations. We are currently conducting biophysical studies to better understand this unique property of our inhibitors. We next aimed to identify an adenosine responsive gene signature in cytotoxic T-cells. RNA sequencing analysis of primary anti-CD3/28/2 activated CD8+ T-cells from human peripheral blood mononuclear cells revealed a unique set of genes persistently and similarly altered in response to both adenosine and AMP. Finally, we demonstrated that CD73 inhibitors can completely block gene expression changes of this AMP/adenosine-response signature. Discovery of an AMP/adenosine-response gene signature may help identify patients whose tumors harbor elevated adenosine signaling and would benefit from an orally bioavailable small molecule inhibitor of CD73 to reverse immunosuppression. Citation Format: Tatiana Zavorotinskaya, Brian Blank, Brenda Chan, Chelsea Chen, Yuping Chen, Xiaohui Du, Frank Duong, Lori Friedman, Tom Huang, Melissa R. Junttila, Wayne Kong, Todd C. Metzger, Jared T. Moore, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Natalie Yuen. CD73 inhibition with a novel orally bioavailable small molecule blocks adenosine production and rescues T-cells activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1023.

Pan proviral insertion site of Moloney murine leukemia (PIM) 1, 2, and 3 kinase inhibitors have recently begun to be tested in humans to assess whether pan PIM kinase inhibition may provide benefit to cancer patients. Herein, the synthesis, in vitro activity, in vivo activity in an acute myeloid leukemia xenograft model, and preclinical profile of the potent and selective pan PIM kinase inhibitor compound 8 (PIM447) are described. Starting from the reported aminopiperidyl pan PIM kinase inhibitor compound 3, a strategy to improve the microsomal stability was pursued resulting in the identification of potent aminocyclohexyl pan PIM inhibitors with high metabolic stability. From this aminocyclohexyl series, compound 8 entered the clinic in 2012 in multiple myeloma patients and is currently in several phase 1 trials of cancer patients with hematological malignancies.

Introduction: CD73 mediates the final dephosphorylation in the conversion of extracellular ATP to adenosine, a metabolite which signals through the A2 family of receptors. Like PD-1, adenosine appears to be part of a negative feedback loop to limit immune activation and prevent excessive inflammation in the context of tissue damage. However, intratumoral hypoxia likely drives excessive CD73-mediated adenosine generation and prevents optimal anti-tumor immune responses. We show that intratumoral adenosine levels are sufficient to drive pervasive suppression of multiple immune subsets in vitro and an orally bioavailable small molecule can prevent adenosine generation in vitro and within the tumor in vivo. Methods We examined the effects of adenosine signaling on multiple immune cell subsets through in vitro functional assays. We interrogated T cell activation, proliferation, and cytolytic activity, as well as NK cell function in vitro. Furthermore, we investigated and identified functional targets of adenosine signaling within T cells using transcriptomic and protein expression analyses. In addition, we performed in vitro myeloid differentiation assays to determine the effect of adenosine signaling on myeloid lineage maturation. Finally, we explored the activity of a novel, orally bioavailable inhibitor of CD73 in vitro and assessed its ability to rescue T cell activation and suppress AMP conversion to adenosine within the tumor microenvironment. Results and Conclusions We found that T cell-expressed CD73 was sufficient to drive adenosine generation from AMP in vitro, resulting in suppression of anti-CD3/CD28-induced T cell activation and proliferation and a reduced capacity to kill cognate antigen-expressing tumor cells. Adenosine-exposed T cells displayed decreased induction of markers associated with activation at both the mRNA and protein level, including Ki67, ICOS, and PD-1, while showing higher expression of naïve-associated CD73. Interestingly, we also observed a third subset of markers which were uniquely induced by adenosine. In addition to its activity on T cells, adenosine signaling caused functional suppression of NK cells in the presence of target Yac-1 cells. In the myeloid compartment, NECA, a stable analog of adenosine, prevented in vitro differentiation of CD103+ cross-presenting dendritic cells, a population which provides a critical stimulus to tumor-infiltrating T cells. Additionally, NECA impaired macrophage expression of CD80, a canonical M1 marker and mediator of T cell costimulation, in both the presence and absence of M1-polarizing IFNγ and LPS. Finally, we showed that a novel, orally bioavailable CD73 inhibitor was able to effectively inhibit AMP to adenosine conversion both in vitro and in vivo, while an anti-CD73 antibody had incomplete effects. Taken together, an orally bioavailable small molecule inhibitor of CD73 represents a potential therapeutic approach to reverse immunosuppression within the tumor microenvironment. Citation Format: Todd C Metzger, Brian R Blank, Brenda Chan, Chelsea Chen, Yuping Chen, Xiaohui Du, Frank L Duong, Valeria R. Fantin, Lori S Friedman, Jared T Moore, Daqing Sun, Jessica Sun, Dena Sutimantanapi, Qiuping Ye, Natalie Yuen, Tatiana Zavorotinskaya. Intratumoral immunosuppression is reversed by blocking adenosine production with an oral inhibitor of CD73 [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-A19. doi:10.1158/1535-7163.TARG-19-LB-A19

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery., PLoS ONE, 12 (4), ISSN:1932-6203

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which is activated by its endogenous steroid hormone ligand cortisol, and by synthetic glucocorticoids such as dexamethasone. Several preclinical studies have shown that GR mediates resistance to both targeted therapies and conventional chemotherapies in a variety of epithelial cancers including prostate, lung, bladder, renal, ovarian and triple-negative breast cancers (TNBC) (Gassler et al., 2005; Li et al., 2017; Zhang et al., 2007). In TNBC, both GR activation and a disrupted cortisol secretion cycle are associated with chemotherapy resistance, increased disease recurrence and poor prognosis (Pan et al., 2011; Skor et al., 2013). Therefore a molecule that inhibits GR activation could attenuate the development of therapy resistance and improve patient outcomes. We have developed novel GR inhibitors that effectively block GR transcriptional activity in cells by competing for ligand binding and by blocking GR-coactivator interactions. In vitro, treatment of TNBC cells with the GR antagonist OP-3713 blocks GR transcriptional activity and enhances the efficacy of chemotherapeutic agents. In rodents the predominant glucocorticoid is corticosterone (Siswanto et al., 2008), which is a weak agonist of human GR. Therefore, to fully activate GR in human xenograft cancer models it is necessary to provide exogenous cortisol. Using xenograft models of TNBC, we find that tumors grown in mice with physiologically relevant circulating cortisol levels to activate GR are significantly less sensitive to chemotherapy than those grown in the absence of cortisol. Furthermore, inhibition of GR by OP-3713 prevents tumor relapse following chemotherapy treatment. We have begun to elucidate the mechanisms by which GR mediates chemoresistance in TNBC, and the basis for the reversal by OP-3713. Our findings underscore the important role of GR as a mediator of resistance in TNBC and highlight the therapeutic potential of GR inhibitors in combination with clinically relevant chemotherapeutic agents. Citation Format: Nadine Jahchan, Haiying Zhou, Wayne Kong, Dan Mc Weeney, Ted Tracy, James Stice, Dena Sutimantanapi, Chelsea Chen, Tom Huang, Yosup Rew, Xiauhui Du, Tatiana Zavorotinskaya, Daqing Sun, Qiuping Ye, Erica Jackson, Valeria Fantin. A novel glucocorticoid receptor (GR) antagonist overcomes GR-mediated chemoresistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1968.

The Pim proteins (1, 2 and 3) are serine/threonine kinases that have been found to be upregulated in many hematological malignancies and solid tumors. As a result of overlapping functions among the three isoforms, inhibition of all three Pim kinases has become an attractive strategy for cancer therapy. Herein we describe our efforts in identifying potent pan-PIM inhibitors that are derived from our previously reported pyridyl carboxamide scaffold as part of a medicinal chemistry strategy to address metabolic stability.

Pan proviral insertion site of Moloney murine leukemia (PIM) 1, 2, and 3 kinase inhibitors have recently begun to be tested in humans to assess whether pan PIM kinase inhibition may provide benefit to cancer patients. Herein, the synthesis, in vitro activity, in vivo activity in an acute myeloid leukemia xenograft model, and preclinical profile of the potent and selective pan PIM kinase inhibitor compound 8 (PIM447) are described. Starting from the reported aminopiperidyl pan PIM kinase inhibitor compound 3, a strategy to improve the microsomal stability was pursued resulting in the identification of potent aminocyclohexyl pan PIM inhibitors with high metabolic stability. From this aminocyclohexyl series, compound 8 entered the clinic in 2012 in multiple myeloma patients and is currently in several phase 1 trials of cancer patients with hematological malignancies.

The clinical manifestations of allergic asthma are believed to result from a dysregulated, T helper 2 lymphocyte (Th2)-biased response to antigen. Although asthma symptoms can be controlled acutely, there is a need for a therapy that will address the underlying immune dysfunction and provide continuous control of chronic airway inflammation. The Th2-type cytokines, IL-13 and IL-4, have been demonstrated to play a crucial role in asthma pathogenesis and their selective neutralization results in the alleviation of asthmatic symptoms in mouse models. The activity of both of these cytokines can be inhibited by a mutant IL-4 protein, IL-4 receptor antagonist (IL-4RA), and thus, continual IL-4RA therapy might be beneficial in treatment of chronic asthma. To explore the potential utility of long-term gene therapy for the treatment of asthma we used a recombinant adeno-associated virus (AAV) vector to deliver and provide sustained expression of IL-4RA in vivo. We show that AAV-mediated delivery of IL-4RA to the airways of mice reduces airway hyperresponsiveness (AHR) and airway eosinophilia triggered by either IL-13 or IL-4. Furthermore, AAV-delivered IL-4RA, expressed either systemically or in the airways of mice following allergen sensitization, significantly inhibited development of airway eosinophilia and mucus production and reduced the levels of asthma-associated Th2 cytokines and AHR in the experimental mouse model of allergic asthma. Thus, gene therapy can be a potential therapeutic option to treat and control chronic airway inflammation and asthmatic symptoms.

In the receptor for ecotropic murine leukemia viruses, tyrosine 235 contributes a critical hydrophobic side chain to the virus-receptor interaction (14). Here we report that tryptophan 142 in ecotropic Moloney murine leukemia virus envelope protein is essential to virus binding and infection. Replacement of tryptophan 142 by alanine or serine resulted in misfolding. However, replacement by methionine (W142M) allowed correct folding of the majority of glycoprotein molecules. W142M virus showed a marked reduction in virus binding and was almost noninfectious, suggesting that tryptophan 142 is involved in receptor binding. In contrast, W142Y virus containing a replacement of tryptophan 142 with an aromatic residue (tyrosine) was as efficient as wild-type virus in infection and binding of cells expressing the wild-type receptor. However, W142Y virus was 100-fold less efficient than wild-type virus in infection of cells expressing a mutant receptor containing tryptophan instead of the critical tyrosine. These results strongly support tryptophan 142 being an essential residue on the virus envelope protein that interacts directly with the critical hydrophobic residue at position 235 of the ecotropic receptor. Tryptophan 142 forms one side of a shallow hydrophobic pocket on the surface of the envelope protein, suggesting that it might comprise the complete putative binding site for tyrosine 235. We discuss the implications of our findings with respect to two models of the envelope protein trimer. Interestingly, both models place tryptophan 142 at the interface between adjacent subunits of the trimer.

It is thought that complete cleavage of retroviral envelope protein into mature surface protein (SU) and transmembrane protein (TM) is critical for its assembly into virions and the formation of infectious virus particles. Here we report the identification of highly infectious, cleavage-deficient envelope mutant proteins. Substitution of aspartate for lysine 104, arginines 124 and 126, or arginines 223 and 225 strongly suppressed cleavage of the envelope precursor and yet allowed efficient incorporation of precursor molecules as the predominant species in virions that were almost as infectious as the wild-type virus. These results indicate that cleavage of the envelope precursor into mature SU and TM is not necessary for assembly into virions. Moreover, they call into question how many mature envelope protein subunits are required to complete virus entry, suggesting that a very few molecules suffice. The failure of host cell proteases to cleave these mutant proteins, whose substitutions are distal to the actual site of cleavage, suggests that the envelope precursor is misfolded, sequestering the cleavage site. In agreement with this, all cleavage mutant proteins exhibited significant losses of receptor binding, suggesting that these residues play roles in proper envelope protein folding. We also identified a charged residue, arginine 102, whose substitution suppressed envelope cleavage and allowed precursor incorporation but resulted in virions that were virtually noninfectious and that exhibited the greatest reduction in receptor binding. Placement of these cleavage mutations into envelope proteins of targeted retroviral vectors for human gene therapy may prevent loss of the modified surface proteins from virions, improving their infectivity and storage hardiness.

Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein—histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.

Purpose: PIM kinases have been shown to act as oncogenes in mice, with each family member being able to drive progression of hematologic cancers. Consistent with this, we found that PIMs are highly expressed in human hematologic cancers and show that each isoform has a distinct expression pattern among disease subtypes. This suggests that inhibitors of all three PIMs would be effective in treating multiple hematologic malignancies. Experimental Design: Pan-PIM inhibitors have proven difficult to develop because PIM2 has a low Km for ATP and, thus, requires a very potent inhibitor to effectively block the kinase activity at the ATP levels in cells. We developed a potent and specific pan-PIM inhibitor, LGB321, which is active on PIM2 in the cellular context. Results: LGB321 is active on PIM2-dependent multiple myeloma cell lines, where it inhibits proliferation, mTOR-C1 signaling and phosphorylation of BAD. Broad cancer cell line profiling of LGB321 demonstrates limited activity in cell lines derived from solid tumors. In contrast, significant activity in cell lines derived from diverse hematological lineages was observed, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma and non-Hodgkin lymphoma (NHL). Furthermore, we demonstrate LGB321 activity in the KG-1 AML xenograft model, in which modulation of pharmacodynamics markers is predictive of efficacy. Finally, we demonstrate that LGB321 synergizes with cytarabine in this model. Conclusions: We have developed a potent and selective pan-PIM inhibitor with single-agent antiproliferative activity and show that it synergizes with cytarabine in an AML xenograft model. Our results strongly support the development of Pan-PIM inhibitors to treat hematologic malignancies. Clin Cancer Res; 20(7); 1834–45. ©2014 AACR.

Proviral insertion of Moloney virus (PIM) 1, 2, and 3 kinases are serine/threonine kinases that normally function in survival and proliferation of hematopoietic cells. As high expression of PIM1, 2, and 3 is frequently observed in many human malignancies, including multiple myeloma, non-Hodgkins lymphoma, and myeloid leukemias, there is interest in determining whether selective PIM inhibition can improve outcomes of these human cancers. Herein, we describe our efforts toward this goal. The structure guided optimization of a singleton high throughput screening hit in which the potency against all three PIM isoforms was increased >10,000-fold to yield compounds with pan PIM Kis < 10 pM, nanomolar cellular potency, and in vivo activity in an acute myeloid leukemia Pim-dependent tumor model is described.

Multiple myeloma (MM) is the second most common hematologic malignancy. Despite recent treatment advances, it remains incurable. Here, we report that Pim2 kinase expression is highly elevated in MM cells and demonstrate that it is required for MM cell proliferation. Functional interference of Pim2 activity either by short hairpin RNAs or by a potent and selective small-molecule inhibitor leads to significant inhibition of MM cell proliferation. Pim inhibition results in a significant decrease of mammalian target of rapamycin C1 (mTOR-C1) activity, which is critical for cell proliferation. We identify TSC2, a negative regulator of mTOR-C1, as a novel Pim2 substrate and show that Pim2 directly phosphorylates TSC2 on Ser-1798 and relieves the suppression of TSC2 on mTOR-C1. These findings support Pim2 as a promising therapeutic target for MM and define a novel Pim2-TSC2-mTOR-C1 pathway that drives MM proliferation.

Efforts to achieve cell type-specific transduction of retroviral vectors for gene therapy have centred on modification of the envelope protein (Env). Typically, addition of a ligand to Env gives binding to the new or target receptor, but little or no infection, and affects the subunit association of the modified Env. We previously discovered two point mutations that increase targeted infection by over 1000-fold when added to an Env modified by N-terminal insertion of the receptor-binding domain from amphotropic murine leukemia virus Env. Here, we asked whether these mutations would similarly increase transduction by Env modified with a clinically relevant ligand, human interleukin-13 (IL-13L). Addition of the point mutations stabilized the weak subunit association observed in some IL-13L-modified Env proteins, but infection via the target IL-13 receptor still did not occur. Fluorescence-based cell–cell fusion assays and studies with a membrane-curving agent revealed that defects in membrane fusion differed with the site of ligand insertion. When IL-13 was inserted into the N terminus of Env, membrane fusion was blocked prior to membrane-lipid mixing, regardless of whether flanking flexible linkers were added. Unexpectedly, insertion of IL-13 in the proline-rich region showed evidence of initiation of fusion and fusion-peptide exposure, but fusion was blocked at a subsequent step prior to fusion-pore formation. Thus, the site of ligand insertion influenced initiation of membrane fusion and its progression. These observations suggest that a novel site for ligand insertion must be identified before clinically useful targeted transduction will be achieved.

The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses. However, changes in a conserved SPHQ motif near the N terminus of the receptor binding subunit of a murine leukemia virus (MLV) envelope protein block infection and induction of cell-cell fusion but not receptor binding. Here we report evidence that a histidine-to-arginine change at position 8 (H8R) in the SPHQ motif of Moloney MLV blocks infection by arresting virus-cell fusion at the hemifusion state. In cell-cell fusion assays, H8R envelope protein induced mixing of membrane outer leaflet lipids but did not lead to content mixing, a finding indicative of fusion pore formation. Kinetic studies of virus-cell fusion showed that lipid mixing of H8R virus membranes begins much later than for wild-type virus. The length of the delay in lipid mixing decreased upon addition of two second-site changes that increase H8R virus infection to 100-fold less than the wild-type virus. Finally, chlorpromazine, dibucaine, and trifluoperazine, agents that induce pores in an arrested hemifusion state, rescued infection by H8R virus to within 2.5-fold of the level of wild-type virus infection and cell-cell fusion to half that mediated by wild-type envelope protein. We interpret these results to indicate that fusion progressed to the hemifusion intermediate but fusion pore formation was inhibited. These results establish that membrane fusion of Moloney MLV occurs via a hemifusion intermediate. We also interpret these findings as evidence that histidine 8 is a key switch-point residue between the receptor-induced conformation changes that expose fusion peptide and those that lead to six-helix bundle formation.

Mutated forms of KRAS are no longer able to switch effectors between “on” and “off” states. It is known that the function of KRAS is controlled by key parts in the C-terminus, including six consecutive lysines, a terminal prenyl moiety and a terminal carboxymethyl functional group. We set out to discover compounds which would inhibit the function of mutated KRAS as an activator for effectors. This campaign yielded several compounds that blocked biochemical and cellular functions of KRAS with low micromolar activity while not affecting markers outside of KRAS pathways in cells. In order to understand the mode of binding of these compounds to KRAS, we generated different forms of the protein, including unprenylated truncated and fully processed full-length protein. NMR studies with truncated protein (amino acids 1-169) identified a site at which compound binding stabilized the inactive conformation of KRAS. This site is located adjacent to switch-II and is similar to sites described by others. The Kd determined for this binding event is almost 3 orders of magnitude higher than the IC50 and EC50 values measured in biochemical and cellular assays. In order to understand this difference, we developed a biophysical assay using the Fortebio system which enabled binding studies in a system with full-length prenylated protein in the presence of lipids, to match the context of the biochemical and cellular assays. Micromolar binding to the full-length prenylated KRAS protein was observed in the Fortebio assay and binding was not observed in the absence of prenylation, consistent with the near millimolar Kd observed by NMR for truncated KRAS. Curiously, similar micromolar binding was seen to a peptide derived from the C-terminus of KRAS (amino acids 168-185) with and without prenyl modification while related compounds that do not bind to the full-length prenylated KRAS also do not bind to these peptides. It is still unclear whether binding to the terminal peptide in lipid context is related to the binding site adjacent to switch-II. From a drug discovery perspective, it remains to be confirmed whether current inhibitors can be optimized. Citation Format: Johanna Jansen, Wolfgang Jahnke, Susan Fong, Laura Tandeske, Charles Wartchow, Keith Pfister, Tatiana Zavorotinskaya, Anke Blechschmidt, Dirksen Bussiere, Yumin Dai, Jeff Dove, Eric Fang, David Farley, Jean-Michel Florent, John Fuller, Simona Gokhin, Alvar Gossert, Mohammad Hekmat-Nejad, Chrystèle Henry, Julia Klopp, Bill Lenahan, Andreas Lingel, Arndt Meyer, Jamie Narberes, Gwynn Pardee, C Gregory Paris, Savithri Ramurthy, Paul Renhowe, Sebastien Rieffel, Kevin Shoemaker, Sharadha Subramanian, Tiffany Tsang, Stephania Widger, Armin Widmer, Isabel Zaror, Stephen Hardy. Inhibiting mutated KRAS, a broken switch of effector pathways. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B38. doi: 10.1158/1557-3125.RASONC14-B38

The MAPK signaling cascade, comprised of the RAS GTPases, the RAF, MEK1/2 and ERK1/2 kinases is frequently deregulated in cancer. ERK1 and ERK2 transmit signals generated by mutant BRAF, Ras or by activated receptor tyrosine kinases to a wide range of nuclear and cytoplasmic substrates, resulting in signal amplification, cell growth, migration and survival. ERK1 and ERK2 have been considered as redundant because of their high homology, large number of overlapping substrates, and ability to substitute for each other in genetically engineered mouse models. Nevertheless, several investigators have identified non-redundant roles for ERK isoforms in oncogenesis; for instance, ERK2, but not ERK1, appears to be responsible for RASmut induced epithelial-to-mesenchymal transformation. Besides, each of the ERK isoforms employs spatially distinct substrate docking domains, DEF (docking site for ERK FXFP) and D (docking domain), to signal to different subsets of substrates and differentially transmit signals downstream. We set out to determine the roles of ERK isoforms as well as DEF- and D-domain dependent signaling in the survival of melanoma tumor cells expressing activating BRAF mutations which are highly sensitive to pharmacological inhibitors of RAF, MEK1/2 and ERK1/2. We designed ERK1 and ERK2 mutants resistant to ATP-competitive ERK1/2 inhibitors and employed auto-activating, MEK-independent, ERK1 and ERK2 mutants to ask if BRAFmut melanoma survival is dependent on either or both ERK isoforms. In addition, we used RNAi and zinc-finger nucleases’ to knockdown or delete each of ERK isoforms. These experimental approaches consistently demonstrated that ERK2, but not ERK1, was the sole driver of cell survival in multiple BRAFmut melanoma cell lines. Moreover, genome-wide gene expression analysis indicated that ERK2, but not ERK1, was largely responsible for transcriptional effects imposed by pharmacological RAF, MEK1/2 or ERK1/2 inhibitors. Thus, in BRAFmut melanoma, functions of ERK1 and 2 are not redundant, and ERK1 cannot substitute for a disabled ERK2. Next, we introduced DEF- and D-substrate docking domain mutations into an ERK inhibitor resistant ERK2 to investigate whether signaling through either domain is sufficient to support melanoma survival. We observed that signaling through D- or DEF- domains of ERK2 had differential effects on gene expression and substrate phosphorylation. Consequently, we have found that a subset of melanoma cell lines was sensitive to elimination of DEF- docking domain interactions, whereas another subset of cell lines tolerated mutations in the DEF-site. Interactions and signaling through ERK D-docking site were dispensable for survival of all melanoma cell lines tested. These data suggest potential novel approaches to target oncogenic MAPK pathway. Citation Format: Tatiana Zavorotinskaya, Upasana Mehra, Yumin Dai, Michel Faure, Ken Crawford, Karen Yu, Jan Marie Cheng, Xiaolei Ma, Jan Xuan, Kelly Yan, Mohammad Hekmat-Nejad, Hanne Merritt, Darrin Stuart, Charles Voliva. Dissecting MAPK pathway in BRAFmut melanoma: Intricacies of ERK1 and ERK2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-121. doi:10.1158/1538-7445.AM2014-LB-121

The current strategy of targeting retroviral vector transduction by inserting a peptide ligand into the envelope protein has met with several obstacles. These modified proteins redirected vector binding to a new cognate receptor on a specific cell type but gave little or no gene transfer because they did not fuse the vector and target cell membranes. They dissociated readily from vectors and often required coassembly of wild-type envelope protein. Here we report a novel strategy to overcome the fusion and stability defects of modified retroviral envelope proteins. We inserted a prototypic ligand, the receptor binding domain of amphotropic murine leukemia virus, into an ecotropic murine leukemia virus envelope protein mutant containing glutamine 227-to-arginine plus aspartate 243-to-tyrosine substitutions. This modified protein increased transduction redirected to human cells expressing the amphotropic receptor to a level within 10-fold that of wild-type amphotropic virus, an increase of as great as 2000-fold over transduction by modified protein lacking the mutations. In addition to suppressing the fusion defect, these mutations unexpectedly stabilized the association of the modified protein with vector particles. Insertion of clinically relevant ligands into this envelope mutant should improve the efficiency and reliability of retroviral transduction of specific cell types for gene therapy applications.

Background PIM1, PIM2, and PIM3 are a closely related family of constitutively active serine/threonine kinases. PIM kinases were first identified as common integration sites in MMLV-induced murine T-cell lymphomas, with these viral insertions resulting in transcriptional activation and expression of the kinase. Similar unbiased insertional mutagenesis screens were used to demonstrate that the three kinases could substitute for each other in this oncogenic process, indicating that inhibition of all three PIM kinases may be necessary to achieve a therapeutic benefit. In human cancers, increased expression and activity of PIM kinases has been detected in many hematopoietic malignancies and solid tumors (prostate, lung, liver) and it is thought that this contributes to cancer cell survival and proliferation in these malignancies. Methods & Results We have developed a potent and specific pan-PIM inhibitor, LGH447, and shown that it is active in PIM2-dependent Multiple Myeloma (MM) cell lines by inhibiting proliferation, mTOR-C1 signaling and phosphorylation of BAD. In addition, LGH447 inhibited proliferation of several AML cell lines. Furthermore, we demonstrated that LGH447 inhibits tumor growth in several mouse subcutaneous xenograft models of MM and AML, where modulation of the pharmacodynamic marker phospho-S6 Ribosomal Protein was used to predict efficacy. We have also determined that LGH447 significantly reduced the bone tumor burden in a disseminated orthotopic human xenograft model of MM. Finally, we have demonstrated increased activity of LGH447 in combination with the PI3K inhibitor BYL719 in a MM model and with Cytarabine in an AML model. Summary Our Results have supported the clinical development of LGH447, a Pan-PIM inhibitor, in relapsed, refractory MM, and further suggest that it may be effective in the treatment of AML and other hematological malignancies, either as a single agent or in combination with other select therapeutic agents. Disclosures: Garcia: Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Langowski:Novartis Institutes for Biomedical Research: Employment. Holash:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Burger:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Zang:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Zavorotinskaya:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Fanton:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Saci:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Growney:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Vanasse:Novartis Pharmaceuticals Corporation: Employment.

Multiple Myeloma (MM) is the second most common hematological malignancy. Despite the advances of therapeutic regimens over the last decade, nearly all patients eventually relapse, highlighting the need for additional therapeutic agents. Pim kinases, a family of serine/threonine kinase, are reported to be oncogenic and required for the maintenance of hematological malignancies. Here, consistent with previous reports, we show that Pim2 kinase expression is most highly elevated in MM. Importantly, shRNA knockdown of Pim2 leads to a significant inhibition of MM cell growth, suggesting that Pim2 is critical in maintaining MM malignancy. We developed a potent and specific small molecule inhibitor for the Pim kinases. Inhibition of Pim2 by this molecule suppresses MM cell growth. We find that Pim inhibitor treatment results in the suppression of cell proliferation, but not induction of apoptosis, in all MM cell lines tested. Further, we identified mTORC1 signaling, which is critical for cell proliferation, as significantly repressed by Pim2 inhibition. The two major mTORC1 downstream effectors: P70S6K and 4EBP1 are rapidly dephosphorylated upon Pim inhibitor treatment of MM cells. We are currently investigating the potential signaling modules that may mediate Pim2’s regulation on mTORC1 pathway. Taken together, our findings strongly support Pim2 as a promising therapeutic target in MM, given its distinct expression in MM and essential role in maintaining proliferation. Citation Format: Jing Lu, Tatiana Zavorotinskaya, Yumin Dai, Xiaohong Niu, Joseph Castillo, Janet Sim, Jianjun Yu, Kevin Shannon, Pablo Garcia. Pim2 is required for maintaining Multiple Myeloma cell proliferation through modulating mTORC1 pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5194. doi:10.1158/1538-7445.AM2013-5194

Two residues, tyrosine 235 and glutamic acid 237, of the ecotropic murine leukemia virus receptor (ATRC1) have been shown to be essential for receptor-mediated virus envelope binding and entry. We performed genetic analyses to examine the biochemical contribution of these residues in a productive virus-receptor interaction. Altered ATRC1 receptors bearing either a phenylalanine, a tryptophan, a histidine, or a methionine at position 235 mediated ecotropic virus entry comparable to that mediated by ATRC1. In contrast, altered ATRC1 receptors bearing alanine, threonine, serine, or proline at position 235 exhibited a 300- to 10,000-fold decrease in receptor capability. Furthermore, substitution of tyrosine or phenylalanine into the corresponding position (242) of the homologous human protein that lacks ecotropic virus receptor capability resulted in acquisition of ecotropic virus receptor function comparable to that of ATRC1. Substitution of a tryptophan or a histidine at that position of the human protein, however, resulted in a much-reduced receptor capability, suggesting a preference for a benzene ring in the hydrophobic side chain. A similar analysis of proteins substituted at position 237 revealed that aspartic acid, but not arginine or lysine, can functionally substitute for glutamic acid 237 in ATRC1 or at the corresponding position in the human protein. These results suggest a requirement for an acidic and a nearby hydrophobic amino acid for efficient ecotropic virus entry. Similar motifs have been identified in the virus binding sites of other retrovirus receptors, suggesting that the initial step of retrovirus entry may be governed by a common mechanism.