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Mutations in the androgen receptor (AR) ligand-binding domain (LBD) can cause resistance to drugs used to treat prostate cancer. Commonly found mutations include L702H, W742C, H875Y, F877L and T878A, while the F877L mutation can convert second-generation antagonists such as enzalutamide and apalutamide into agonists. However, pruxelutamide, another second-generation AR antagonist, has no agonist activity with the F877L and F877L/T878A mutants and instead maintains its inhibitory activity against them. Here, it is shown that the quadruple mutation L702H/H875Y/F877L/T878A increases the soluble expression of AR LBD in complex with pruxelutamide in Escherichia coli. The crystal structure of the quadruple mutant in complex with the agonist dihydrotestosterone (DHT) reveals a partially open conformation of the AR LBD due to conformational changes in the loop connecting helices H11 and H12 (the H11–H12 loop) and Leu881. This partially open conformation creates a larger ligand-binding site for AR. Additional structural studies suggest that both the L702H and F877L mutations are important for conformational changes. This structural variability in the AR LBD could affect ligand binding as well as the resistance to antagonists.

Purpose To evaluate GT0918, a 2nd-generation AR antagonist, for its AR down-regulation activity among breast cancer patients. Methods The effect of GT0918 on AR protein expression was evaluated in AR expression breast cancer cells and in breast cancer xenograft model. A 3 + 3 phase I dose-escalation study was launched in Peking University Cancer Hospital. The endpoints included dose finding, safety, pharmacokinetics, and antitumor activity. Results GT0918 was demonstrated to effectively suppress the expression of AR protein and the growth of AR-positive breast cancer tumors in mouse xenograft tumor models. All patients treated with GT0918 were at a QD dose-escalation of five dose levels from 100 to 500 mg. The most common treatment-related AEs of any grade were asthenia, anemia, decreased appetite, increased blood cholesterol, increased blood triglycerides, decreased white blood cell count, and increased low-density lipoprotein. Grade 3 AEs were fatigue (2 of 18, 11.1%), aspartate aminotransferase increase (1 of 18, 5.6%), alanine aminotransferase increase (1 of 18, 5.6%), and neutrophil count decrease (1 of 18, 5.6%). Clinical benefit rate (CBR) in 16 weeks was 23.1% (3/13). Among 7 AR-positive patients, 6 can evaluate efficacy, and 2 completed 23.5- and 25-cycle treatment, respectively (as of 2020/1/20). PK parameters showed a fast absorption profile of GT0918 in the single-dose study. GT0918 and its major metabolite reached steady-state serum concentration levels at day 21 after multiple dosing. Conclusion GT0918 can effectively inhibit AR-positive breast cancer tumor growth. GT0918 was demonstrated well tolerated with a favorable PK profile. The suitable dose of GT0918 was 500 mg QD and may provide clinical benefits for AR-positive mBC.

Coronavirus disease 2019 (COVID-19) has caused the public health crisis in the whole world. Anti-androgens block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and protect against severe clinical COVID-19 outcomes. GT0918, a novel androgen receptor antagonist, accelerated viral clearance and increased recovery rate in outpatients by blocking SARS-CoV-2 infection though down-regulating ACE2 and TMPRSS2 expression. Further clinical study showed that GT0918 reduced mortality rate and shortened hospital stay in hospitalized COVID-19 patients. GT0918 also exhibits protective efficacy in severe COVID-19 patient in critical care. However, the mechanism of GT0918 treatment for severe COVID-19 disease is unknown. Here, we found GT0918 decreased the expression and secretion of proinflammatory cytokines through NF-κB signaling pathway. The acute lung injury induced by LPS or Poly(I:C) was also attenuated in GT0918-treated mice, compared with vehicle control group. Moreover, GT0918 elevated the NRF2 protein level but not mRNA transcription activity. GT0918 induced proinflammatory cytokines downregulation was partially dependent on NRF2. In conclusion, our data demonstrate that GT0918 reduced cytokine release and suppressed inflammatory responses through inhibiting NF-κB signaling and activating NRF2. GT0918 is not only effective for treatment of mild to moderate COVID-19 patients, but also a potential therapeutic drug for severe COVID-19 patients by reducing the risk of cytokine storm and acute respiratory distress syndrome.

The oncoprotein c-MYC, a major regulator of the epigenome and transcriptome, is dysregulated in 70% of all human cancers. MYC is highly expressed in Burkitt lymphoma and TP53 mutant and venetoclax (ven) resistant AML (Sallman, Blood 2021, Nishida, ASH 2021). However, targeting c-Myc or the MYC pathway has not been successful and remains a major unmet clinical need. We developed the first cereblon E3 ligase modulators (CELMoDs) for c-MYC: GT19630 and GT19715 (salt form of GT19630). C-MYC was one of the top decreased proteins in chromatin-enriched proteomics, was pulled down by biotinylated GT19630 in in vitro affinity purification assay and was degraded with IC50 of 0.33 nM in MYC-amplified HL-60 cells. Proteasome inhibitor ixazomib completely blocked c-MYC reduction, suggesting a CRL4CRBN-dependent degradation. Blood cancer cell lines responded to GT19715 greater than other cancer cell lines such as lung, breast and brain tumors in a broad cell line panel, providing rationale to develop the degrader in hematologic malignancies. In agreement with other CELMoDs, proteomic analyses revealed degradation of translation termination factor GSPT1 (G1 to S phase transition proteins 1), an important factor in LSC survival Whereas a selective GSPT1 degrader CC-90009 reduced GSPT1 protein levels but not c-MYC, GT19715 reduced both c-MYC and GSPT1 and exerted a 20x higher cytoreduction than CC-90009 (IC50 of 1.8 nM vs 40.4 nM for GT19715 and CC-90009, respectively) in HL-60 cells. GT19630 degraded c-MYC and GSPT1 and inhibited tumor growth in a xenograft model with HL-60 cells. GT19715 eliminated circulating blasts and prolonged survival in the systemic Burkitt lymphoma model (Daudi). GT19715 significantly reduced human CD45+ AML blasts in peripheral blood, bone marrow (BM) and spleens compared to vehicle controls in vivo in a chemotherapy-resistant AML PDX model. MV4;11 venetoclax resistant (VR) cells demonstrated elevated protein levels of c-MYC and GSPT1 and GT19715 induced 4log10 cytoreduction in BM and prolonged survival of mice with MV4;11 VR cells. Baseline c-Myc protein levels associated with sensitivity to GT19715 in MOLM-13 cells with CRISPR engineered knockout/mutations of TP53 (R2 = 0.86, P = 0.02). GT19715 induced comparable cell death in primary AML samples with wild-type or mutant TP53 (95.4% and 91.7% cytoreduction, P = 0.48 for wild-type and mutant TP53 samples at 64 nM of GT19715, respectively). CD34+ AML cells were more susceptible to GT19715 than CD34- AML cells, suggesting a greater efficacy in AML stem/progenitor than in more mature AML cells. Notably, single cell mass cytometry revealed that CD34+ AML cells had higher c-MYC protein levels than CD34+ normal BM cells and GT19715 reduced c-Myc levels in CD34+ AML but not normal BM cells. GT19715 induced greater cytoreduction in CD34+ AML than in normal BM cells, suggesting a therapeutic window. Conclusions: The novel dual c-MYC/GSPT1 degrader GT19715 exerts TP53 independent preclinical anti-lymphoma and -leukemia efficacy, providing rationale for its clinical development. Citation Format: Yuki Nishida, Darah A Scruggs, Edward Ayoub, Lauren B Ostermann, Tallie Patsilevas, Vivian R Ruvolo, Po Yee Mak, Bing Z. Carter, Steffen Boettcher, Abhishek Maiti, Koji Sasaki, Qianxiang Zhou, Zhaohui Yang, Honghua Yan, Liandong Ma, Michael Andreeff. C-MYC Targeting by Degradation: Novel Dual c-MYC/GSPT1 Degrader GT19715 Induces TP53-independent Cell Death in MYC-amplified Acute Myeloid Leukemia and Lymphomas [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A31.

Acute Myeloid Leukemia (AML) is the third hematological malignancies with the worst relative overall 5-year survival rate (11.7%) in hematological malignancies. AML is a heterogeneous disease with a broad spectrum of genomic changes and molecular mutations that lead to a poor prognosis and clinical outcome. Leukemic stem cells progress to myoblasts that continue to proliferate without differentiating, namely, immature blasts in AML. The hedgehog (HH)/glioma-associated oncogene homolog (GLI) signaling pathway is essential for embryonic and stem cell developments. This pathway has been one of the most promising targets for drug discovery and developments for AML. Although HH inhibitor Glasdegib in combo with low-dose cytarabine achieved FDA approval for AML, Venetoclax (BCL-2 inhibitor/ABT-199) plus a hypomethylating agent (HMA) have been dominating the regimens in AML recently. Here we reported GT1708, a HH inhibitor, improves ABT-199 (venetoclax)-induced apoptosis by down-regulating MCL-1 proteins in AML cells. GT1708 is a potent HH inhibitor (IC50=0.11 nM in HH pathway-driven cellular assay) and inhibited GLI expression with doses of 1, 3 and 10 mpk in a HH-dependent medulloblastoma animal models. In Molm-13 (AML) cells, GT1708 was shown to down-regulate the expression of MCL-1 proteins (anti-apoptotic proteins). In contrast, ABT-199 increased the expression of MCL-1. Furthermore, ABT+Aza (Azacidine/HMA drug) induced more MCL-1 expression than ABT-199. Importantly, GT1708 was shown to induce the expression of cleaved-PARP (c-PARP/apoptotic marker) and to increase c-PARP expression when combined with ABT-199. Due to ABT+Aza induced MCL-1 overexpression, the combination of both agents failed to induce c-PARP, suggesting MCL-1 overexpression conferring resistance to ABT+Aza therapy in Molm-13 cells. GT1708 was further evaluated in flow cytometry-based apoptotic assays. GT1708, ABT-199 and Aza were demonstrated to induce an early apoptosis by 9.98%, 47.5%, and 8.64%, respectively. In comparation, ABT+GT1708 were revealed to induce a 61% of apoptosis superior to 47% or 48.5% of apoptosis induced by either ABT or ABT+Aza in Molm-13 cells. These results confirm the role of MCL-1 overexpression in conferring resistance to ABT+Aza therapy, which can be overcome by ABT+GT1708 combo. GT1708/ABT combo were also shown a marginable superior antitumor activity than ABT along in Molm-13 animal models. GT1708 has been testing in a phase I study in AML patients with previous multiple lines of regimens. GT1708 has been shown to reduce blast counts in three of 13 AML patients treated with higher doses and demonstrated favorite PK and safety profiles. In brief, these results support the clinical development of GT1708 in combination with ABT-199 in AML patients. Citation Format: Liandong Ma, Qianxiang Zhou, Honghua Yan, Min Dong, Xiahe Han, Jiangwie Li, Jie Qu, Weidong Qian, Youzhi Tong. Combination of a clinical stage-hedgehog inhibitor, GT1708, improves Venetoclax-induced apoptosis by down-regulating MCL-1 proteins in AML cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4978.

Myc family members (MYC, MYCN and MYCL) are oncogenic transcriptional factors, which form dimers with Max to activate transcription activities to drive tumor initiation and progression. c-Myc deregulation has been identified in 80% of all tumor types, including B-cell malignancies, AML and a variety of solid tumors. N-Myc genetic alterations have been identified in small cell lung cancer, neuroblastoma, neuroendocrine prostate cancers and sarcoma. Myc-deregulation has been directly linked to the poor clinical outcome in these cancers, which makes Myc a therapeutic target for pharmacological inhibition. Here we described GT19630, a c-Myc/n-Myc degrader. GT19630 selectively degraded c-Myc proteins in c-Myc dependent blood cancer cells (IC50=1.5 nM) as compared to growth-factor regulated c-Myc in hematopoietic progenitor cells (TF-1) (IC50=52.5 nM). Similar selectivity of GT19630 has been demonstrated in cell proliferation and granulocyte–macrophage progenitor colony forming unit (GM-CFU) assays with the IC50s of 26.2 and 39.0 nM, respectively. GT19630 was shown to degrade c-Myc via proteasome degradation system and degraded CRBN-dependent endogenous neo-substrates of GSPT1, CK1α and IKZF1. GT19630 reduced transcriptional factors including c-Myc, Max, MXI1, SWF/SNF family members/associated proteins; ARID1A, SMARCE1, and SMARCC1 in transcriptional factor response element (TFRE) assays. Moreover, GT19630 inhibited the cell proliferation with IC50 Citation Format: Liandong Ma, Yuzhi Tong, Zhaohui Yang, Qianxiang Zhou, Honghua Yan, Ye Chen, Dong Chen, Ru Xu, Yini Wang, Jun Qin. Discovery and evaluation of GT19630, a c-Myc/n-Myc degrader, for targeting c-Myc-driven B-cell malignancies, acute myeloid leukemia (AML) and n-Myc driven cancers [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A03.

c-Myc is an oncogenic transcriptional factor driving tumor initiation, progression and poor prognosis in 80% of all tumor types, especially in B-cell malignancies and small cell lung cancer (SCLC) with Myc genomic alterations. Myc dysregulation have been directly linked to the poor clinical outcome in these cancers. Therefore, it is highly warranted to discover and develop novel Myc therapeutical agents for targeting Myc driven cancers. Here we described GT19630, a GSPT1/Myc CELMoD. GT19630 was discovered through an SAR effort for c-Myc degrader by using c-Myc ELISA and Western blot assays in c-Myc driven HL60 AML cells. GT19630 selectively degraded c-Myc proteins in HL60 cells (IC50=1.5 nM) as compared to growth-factor regulated c-Myc erythroid progenitor cells (TF-1 cells) with IC50=52.5 nM. GT19630 also selectively inhibited HL60 cell proliferation (IC50= 0.33 nM), as compared to its IC50 (26.2 nM) in GM-CSF-TF-1 proliferation, as well as in bone marrow colony-forming cell assays (myeloid=40.2 nM), suggesting>100X selectivity for HL60 cells over normal blood cells. Through the SAR for Myc degrader, GT19630 has been evolved as a leading molecule sharing chemical properties to CELMoDs. Therefore, this compound was further evaluated by proteomics and western blot, GT19630 was confirmed to selective degradation of CELMoD targets, GSPT1/GSPT2 (translation termination factor G1 to S phase transition proteins 1 and 2) with IC90 Citation Format: Liandong Ma, Youzhi Tong, Zhaohui Yang, Qianxiang Zhou, Hongha Yan, Ru Xu, Jie Chen, Jie Pan, Huiyuan Wang, Jiangwei Li, Dong Chen, Xiang Cai, Jie Qu, Yini Wang, Jun Qin, Yuki Nishida, Michael Andreeff, Qilnli Guo, Yuki Nishida, Michael Andreeff. Discovery and evaluation of GT19630, a c-Myc/GSPT1 cereblon E3 ligase modulator (CELMoD), for targeting Myc-driven blood cancers and small cell lung cancers (SCLC) [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 5479.

As of November 2021, there were 21 million confirmed active cases of COVID-19, including 77,016 patients in serious or critical condition (virusncov.com). However, there are no effective oral drugs for the treatment of severe COVID 19 patients. We here discuss the mechanism of action for Proxalutaminde to treat mild, moderate and severe COVID-19 Patients. Cellular entry and infection of SARS-CoV-2 virus are mediated by two key proteins in host cells, angiotensin converting enzyme 2 (ACE2), a host transmembrane protein, providing the binding sites for SARS-CoV-2 on the host cell surface, and transmembrane protease serine 2 protein (TMPRSS2), priming the S protein of SARS-Cov-2 to facilitate the viral entry into the host cells. Both ACE2 and TMPRSS2 proteins are regulated by androgen receptor (AR) signaling. Previously, Proxalutamide has been reported to downregulate the expression of ACE2 and TMPRSS2 in cells derived from prostate, lung cancer and normal lung epithelial cells. In this study, we demonstrate that Proxalutamide inhibited the infection of SARS-COV-2 wild type, alpha and delta variants, with IC50s of 69, 48 and 39 nM, respectively. Moreover, Proxalutamide reduced SARS-COV-2 viral load in outpatients with COVID-19 (82% viral RT-PCR negative rate in active group vs. 31% in placebo group after treatment for 7 days (p-value Severe COVID-19 disease leads to cytokine storm resulting in pulmonary inflammation and extensive damage in lung and other organs. Anti-inflammatory drugs, including Baricitinib and dexamethasone, have shown limited clinical benefit for hospitalized COVID-19 patients. Therefore, more effective drugs are in urgent need for patients suffering from severe COVID-19. Recently, Proxalutamide has been reported to reduce the mortality rate (HR=0.16) and lung injury (by 57%, active drug vs placebo groups) in hospitalized patients with COVID-19 in an IIT phase III study. We presented here the mechanism of action of Proxalutamide for targeting cytokine storm in severe COVID-19 patients. Proxalutamide was demonstrated to activate nuclear factor erythroid 2-related factor 2 (Nrf2) in macrophages, which stimulates the antioxidant response element (ARE) for reducing cytokine storm-induced organ damage in COVID-19. In addition, Proxalutamide inhibited TNF alpha and IL-6 expression and blocked INF gamma signaling by downregulating STAT1 expression in immune cells. Importantly, Proxalutamide reduced inflammatory cells in lungs in a Poly (I:C), pseudoviral induced-lung injury animal models. Further, Proxalutamide decreased C-reactive protein, D-Dimer and improved lymphocyte count, biomarkers for COVID-19 progression in clinical studies. Together, these results provide a strong rationale for the treatment of severe COVID-19 patients with Proxalutamide. Citation Format: Liandong Ma, Youzhi Tong, Qianxiang Zhou, Hongha Yan, Xiaodan Hou, Zhiha Ren, Jiangwei Li, Huiyuan Wang, Weidong Qian, Yu Zhang, Andy Goren, Arul Chinnaiyan. Mechanisms of action (MOA) for proxalutamide, an androgen receptor (AR) antagonist, for the treatment of mild, moderate and severe COVID-19 patients [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 5274.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a worldwide pandemic named coronavirus disease 2019 (COVID-19). The disease progression and mortality rates of patients with COVID-19 are higher in males than in females. Hence, we explored the potential mechanism of the COVID-19 gender disparity and searched for potential alternative therapeutic agents as unmet medical needs for male patients with COVID-19. Methods: We collected clinical data from 1339 COVID-19 patients and analyzed androgen receptor (AR) chromatin binding and expression patterns by ChIP-seq and scRNA-seq data. Moreover, we studied the androgen-induced expression of ACE-2 and TMPRSS2, two key proteins for SARS-CoV-2 cellular entry and infection and the blockage of these two key proteins with our AR antagonist Proxalutamide (GT0918) in normal lung cells, cancer cells derived from prostate and lung cancers. Furthermore, we examined the effect of GT0918 on inducible nitric oxide synthase (iNOS) and Tumor necrosis factor-alpha (TNF-α), the macrophage-activation markers, in mouse macrophage cells. Results: We demonstrated higher rates of disease progression and mortality of male COVID-19 patients. The mechanism of the gender disparity may result from the androgen-AR activation in male patients with COVID-19. We revealed that androgen-AR activation induced the expression of ACE2 and TMPRSS2 under the androgen-dependent condition in cells derived from prostate and lung cancer, which was inhibited by the blockage of AR signaling with AR antagonist GT0918. Importantly, GT0918 also inhibited the expression of macrophage activation markers iNOS and TNF-α, the biomarkers for macrophage polarization. These results support the role of androgen-AR signaling in the disease progression and mortality in male patients with COVID-19.

To date, as of 17 November 2020, there have been 55 million confirmed cases of COVID-19, including 1,4 million deaths globally, as reported to WHO. It is, therefore, very critical to discovery and development of the available treatment options for COVID-19. We here discuss the repurposing 2nd generation androgen receptor antagonist Proxalutaminde to treat COVID19. We analyzed the gender disparity of disease severity and progression in 1339 patients with COVID-19 and investigated the mechanism of gender disparity in male vs female patients with COVID 19. As androgen-androgen receptor pathway may contribute to the difference in severity and disease progression in male and female patients with COVID-19, we used cell lines derived from lung, prostate cancer and normal lung epithelial cells to determine the effect of androgen and androgen receptor antagonist Proxalutamide on the expression of two key proteins for SARS-CoV-2 to infect and enter the host cells. Angiotensin converting enzyme 2 (ACE2), a host transmembrane protein provides the binding sites for SARS-CoV-2 on the host cell surface, transmembrane protease serine 2 (TMPRSS2), a cellular serine protease, prims the S protein of SARS-Cov-2 to facilitate the viral entry into the host cells. As cytokine store plays a major role in the disease progression of COVID-19, we examined the effect of GT0918 on inducible nitric oxide synthase (iNOS) and Tumor necrosis factor-alpha (TNF-α), the macrophage polarization/activation markers, in mouse macrophage cells. In this study, we demonstrated higher rates of disease progression and mortality of male COVID-19 patients than female patients. Furthermore, we revealed that androgen-AR activation induced the expression of ACE2 and TMPRSS2 under the androgen-dependent condition in cells derived from prostate and lung cancer, which was inhibited by the blockage of AR signaling with Proxalutamide. Importantly, Proxalutamide also inhibited the expression of iNOS and TNF-α, the biomarkers for macrophage polarization/activation. These results support the role of androgen-AR signaling in the disease progression and mortality in male patients with COVID-19. We are currently conducting clinical study in COVID-19 patients with Proxalutamide in Brazil (NCT04446429). Citation Format: Liandong Ma, Youzhi Tong, Qiang Guo, Yifeng Zhou, Qianxiang Zhou, Honghua Yan. Repurposing 2nd generation androgen receptor antagonist Proxalutamide to treat COVID-19 [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 711.

c-Myc is an oncogenic transcriptional factor, which form a dimer with Max to activate its transcription activity, driving tumor initiation, progression and poor prognosis in 80% of all tumor types. IGH/Myc genomic translocations have been identified in B-cell lymphoma (15-100%). The amplification of Myc family members, MYC, MYCN, or MYCL have been revealed in 20% patients with small cell lung cancers (SCLC). Therefore It is highly warranted to discover and develop novel c-Myc inhibitors targeting c-Myc dependent tumors (Lymphoma and SCLC) with acceptable therapeutic index. Here we described the discovery of GT19077, a c-Myc/Max PPI small molecule inhibitor, and GT19506, a c-Myc PROTAC, for targeting Myc-dependent tumors. GT-19077 was demonstrated to disrupt c-Myc/Max dimer with an IC50 of 1.0 µM in PPI assays and degraded c-Myc protein with IC50 of 0.34 µM in HL-60 cells measured in c-Myc ELISA assays, which was confirmed by Western Blotting (WB). GT19506 (PROTAC) effectively degraded c-Myc proteins in HL-60 cells with an IC50 of 0.10 µM. The selectivity of both compounds against the physiologic function of c-Myc was determined in growth factor-stimulated hematopoietic progenitor cells by using c-Myc ELISA assays. GT19077 and GT19506 were less potent at degrading c-Myc in growth-factor driven hematopoietic cells with IC50 of 2.36 and 2.40 µM respectively, suggesting a 6-fold and 24-fold selectivity, respectively, which was further confirmed by WB. Further, GT19077 and GT19506 were evaluated in panels of blood cancer and SCLC cell lines carrying Myc genetic alterations, as measured by. GT19077 was shown to selectively inhibit the proliferation of B-cell malignant cells with IGH/Myc genomic translocations in a panel of 14 blood cancer cell lines tested by CellTiter-Glo® Luminescent Cell Viability Assays (CTG). Consistently, GT19506 also selectively inhibited the proliferation of B-cell malignant cells in the panel of 28 blood cancer cell lines tested. Both compounds also inhibited the proliferation of SCLC cells in a panel of 18 SCLC cell lines tested (66% and 22%, respectively). Moreover, GT19077 inhibited c-Myc-driven tumor biology, including G2/M arrest and CD-47 and PD-L1 down-regulation. Finally, GT19077 and GT19506 demonstrated PK dependent c-Myc target engagement in HL-60 and Ramos xenograft tumor models. In conclusion, GT19077 and GT19506 are currently in Lead Optimization Phase, which demonstrate the desired selectivity of targeting Myc dependent B-cell malignancies and Myc-amplified SCLC, but sparing c-Myc in growth factor/cytokine regulated blood progenitor cells. The defined selectivity has enabled the ongoing in vivo efficacy studies, currently GT19077 and GT19506 have being evaluated in in vivo efficacy studies. Citation Format: Liandong Ma, Youzhi Tong, Qianxiang Zhou, Zhaohui Yang, Honghau Yan, Ye Chen, Ruo Xu, Jie Pan, Xiaoqi Gou, Weidong Qian, Jie Chen, Qi Sun, Luhua Lai. Discovery of GT19077, a c-Myc/Max protein-protein Interaction (PPI) small molecule inhibitor, and GT19506 a c-Myc PROTAC molecule, for targeting c-Myc-driven blood cancers and small cell lung cancers [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 1265.

c-Myc is a transcriptional factor and an oncogene driver which plays a major role in tumor initiation, progression and poor prognosis in 80% of all tumor types, especially in c-Myc-additive B-cell and myeloid malignancies. c-Myc's critical role as an oncoprotein makes it an attractive target for pharmacological inhibition. However, c-Myc has been considered as “an undruggable” target since it is an intrinsically disordered protein and regulates key physiological functions in normal cells, which can be a liability for effectively pharmacological intervention. Thus, effective and selective c-Myc targeted molecules are highly warranted, but no such inhibitors are now in clinical testing. c-Myc is dependent on the heterodimerization with Max for the stability and activation of transcription. Here we describes a c-Myc/Max PPI small molecule inhibitor, GT-19077, selectively degrades c-Myc and inhibits the proliferation of c-Myc-addictive B-cell and myeloid malignant cells, but spares c-Myc and the cell proliferation in JAK2-STAT5 dependent erythroblast cells. Through the conformation generation/binding site predication, virtual screening of molecules (>200K), optimization and experimental validation, a leading c-Myc binding molecule series was identified. GT19077 was then discovered and optimized as a leading molecule that exhibited a direct c-Myc binding activity in HL-60 myeloid leukemia cells as shown in Cellular Thermal Shift Assay (CETSA). Importantly, GT19077 selectively degraded c-Myc in HL-60 cells with DC50 of 0.40 µM vs. DC50 of 2.31 µM in GM-CSF-JAK2-STAT5 dependent TF-1 erythroblast cells (>5 fold) as measured by ELISA assays. Consistently, GT19077 also selectively inhibited the proliferation of HL-60 cells with the IC50 of 0.34 µM vs. IC50 of 1.6 µM in GM-CSF stimulated TF-1 cells (>5 fold). Profiling on the proliferation of a selected 14 hematologic malignant cell panel with c-Myc overexpression, GT19077 selectively inhibited the proliferation of all seven B-cell malignant cell lines tested, which include three carrying Myc-IGH translocations and two expressing Kras G12A mutations, with IC50s as low as 160 nM. In addition, two myeloblast cell lines and one monoblast cell line were also sensitive to GT19077 with IC50 of 300-700 nM. In contrast, GT19077 showed almost no effect on the proliferation of four leukemia cell lines with known oncogene drivers, including two erythroblasts (JAK2-STAT5 dependent), one macrophage (FGFR1OP2-FGFR1) and one myeloblast (AML-ETO). These results suggest that c-Myc inhibitor GT19077 can selectively target c-Myc-addictive B-cell and myeloid malignancies, but spares the liability on JAK2-STAT5 driven erythroblasts. Moreover, GT19077 has almost no effect on hematologic malignancies with different oncogene drivers that can be used as biomarkers for patient stratification. Further, GT19077 demonstrated a favorable PK profile with the bioavailability of 57.3% in rat by intraperitoneal (IP) administration. Currently GT19077 is being evaluated in B-cell and myeloid malignant animal efficacy models. Since this novel c-Myc inhibitor demonstrates a superior potency and a unique electivity profile, as compared to many experimental c-Myc inhibitors, it has potential for the drug development for targeting c-Myc-addictive B-cell and myeloid malignancies. Citation Format: Liandong Ma, Youzhi Tong, Zhaohui Yang, Qian Xiang Zhou, Jie Chen, Ye Chen, Honglei Bi, Jie Pan, Qi Sun, Ru Xu, Luhua Lai. GT19077, a c-Myc/Max protein-protein interaction (PPI) small molecule inhibitor, selectively targets c-Myc-additive B-cell and myeloid malignancies [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-111.

186 Background: AR blockade is an important treatment option for mCRPC in clinic and GT0918 is a new chemical entity of AR blocker in 2nd generation. A phase I dose escalation study was planned in pts with mCRPC progressed on multiple lines of SoC and experimental therapies. Daily oral administration of GT0918 has shown better clinical outcomes in 400mg and 500mg cohorts with no comprised toxicities. To study the tumor biology in response to study drug in clinical setting, CTC and cfDNA/RNA based biomarkers were explored. Methods: Pts with histologically confirmed mCRPC who progressed on enza, abi, docetaxel, etc were enrolled and treated with GT0918 continuously until PD, intolerable toxicity or withdraw. Blood samples were collected at baseline, on study drug every 8 wks during the trial and pts with ≥ 3 blood test samples were qualified for various assays for CTCs and cfDNA/RNA via EPIC and PredicinePlus platforms. Results: Total 40 pts were orally administrated GT0918 with dose increasing 50, 100, 200, 300, 400, 500 and 600 mg daily and shown well tolerated with mild to moderate toxicities. Pts received GT0918 over 16 weeks were run biomarkers in Predicine and/or Epic platforms. ctDNA/RNA based variants and CTCs are all detectable in selected pts samples. AR splicing variants (AR-V3 and AR-V7), AR hotspot mutations (W742C, T878A and S889G) and amplifications were detected and shown interesting trends with the clinical outcomes. Both exploratory biomarkers and CTCs suggested higher doses of GT0918 resulted in better clinical outcomes. Conclusions: This is a preliminary study to explore genomic alterations and the CTC enumeration in late stage of mCRPC pts in response to GT0918 treatment with dose increase. As non-invasive assays, both CTC and ctDNA/RNA assays provided valuable molecular insights for monitoring treatment effects besides PSA and imaging scan. Early detection of possible drug sensitivity/resistance mechanisms will facilitate clinical development programs. More patients will be tested in phase II study GT0918 in mCRPC progressed on either abiraterone or enzalutamide. Clinical trial information: NCT02826772. [Table: see text]

Reactivation of androgen receptor (AR) plays a key role in prostate cancer growth, especially in castration-resistant prostate cancer (CRPC) progression. Accumulated data have demonstrated that the AR reactivation is resulted from AR amplification, AR overexpression and mutations in AR ligand binding domain (LBD) as well as AR splicing variants (AR-V7). This AR reactivation also drives the resistance to AR targeted therapies, including 1st and 2nd generations of AR antagonists, such as Bicalutamide (Bica) and Enzalutamide (Enza, MDV3100). Therefore, the development of novel and effective AR antagonists is in urgent need for overcoming AR reactivation for patients with metastatic castration-resistant prostate cancer (mCRPC). Here we report a novel AR binding pose of Proxalutamide (GT0918), an AR antagonist, in silico of drug’s mode of action. We also reveal the effects of GT0918 on the transactivation of the wild type AR (wtAR) and AR mutants with comparisons to 1st and 2nd generations of AR antagonists (Bicalutamide and Enzalutamide). GT0918 was reported to have higher binding affinity than Bica (11.4X) and Enza (3.5X) in AR ligand binding assays previously. To gain detail insights into the mode of action of GT0918 and Enza in AR LBD, structure-based computer modeling was performed. The mode of drug action shows that GT0918 binds to AR LBD pocket. This compound not only fits in the AR LBD pocket very well, but also its structure pushes AR Helix 12 away, resulting in the decreased interaction between AR Helix 12 and LBD pocket, which creates a unique and significant difference from other AR blockers. GT0918 was further assessed in luciferase-based AR transactivation assays. The results showed GT0918 significantly inhibited the androgen-induced transactivation of the wtAR. Importantly, it also blocked the transcriptional activity of tested mutant ARs arising to targeted AR therapies, including the AR mutant F877L that converts the Enzalutamide and Apalutamide (ARN-509) from antagonist to agonist activity. In clinical trial setting, some mCRPC patients progressed on Enzalutamide and bicalutamide showed stable disease and durable responses after received GT0918 from various doses. In conclusion, our results support the clinical development of GT0918 in prostate cancer patients who progressed on Enzalutamide or/and AR blockers. Citation Format: Liandong Ma, Qianxiang Zhou, Yang He, Jie Chen, Karl Zhou, Qingqing Zhou, Chunyun Chen, Jason Shaoyun Xiang, Xiaobing Deng, Luhua Lai, Youzi Tong. Proxalutamide (GT0918), a novel androgen receptor (AR) antagonist, targeting resistance mechanisms to AR signaling-directed castration-resistant prostate cancer (CRPC) therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3861.

The mechanisms of resistance to the antimetabolite gemcitabine in non-small cell lung cancer have not been extensively evaluated. In this study, we report the generation of two gemcitabine-selected non-small cell lung cancer cell lines, H358-G200 and H460-G400. Expression profiling results indicated that there was evidence for changes in the expression of 134 genes in H358-G200 cells compared with its parental line, whereas H460-G400 cells exhibited 233 genes that appeared to be under- or overexpressed compared with H460 cells. However, only the increased expression of ribonucleotide reductase subunit 1 (RRM1), which appeared in both resistant cell lines, met predefined analysis criteria for genes to investigate further. Quantitative PCR analysis demonstrated H358-G200 cells had a greater than 125-fold increase in RRM1 RNA expression. Western blot analysis confirmed high levels of RRM1 protein in this line compared with the gemcitabine-sensitive parent. No significant change in the expression of RRM2 was observed in either cell line, although both gemcitabine-resistant cell lines had an approximate 3-fold increase in p53R2 protein. A partial revertant of H358-G200 cells had reduced levels of RRM1 protein (compared with G200 cells), without observed changes in RRM2 or p53R2. In vitro analyses of ribonucleotide reductase activity demonstrated that despite high levels of RRM1 protein, ribonucleotide reductase activity was not increased in H358-G200 cells when compared with parental cells. The cDNA encoding RRM1 from H358-G200 cells was cloned and sequenced but did not reveal the presence of any mutations. The results from this study indicate that the level of RRM1 may affect gemcitabine response. Furthermore, RRM1 may serve as a biomarker for gemcitabine response.

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.

Background: MET amplification (amp) is a resistance mechanism to EGFR TKI treatment. Emibetuzumab, a bivalent MET antibody (Ab) blocks HGF binding to MET and internalizes the receptor. Combination of emibetuzumab with EGFR TKIs (erlotinib, AZD9291, CO1686) or EGFR Ab (necitumumab, cetuximab) was evaluated in 3 ER xenograft models. Methods: Model 1: ER cell line HCC827ERL with high focal MET amp, high pMET, EGFR ex19 del (no T790M) was created from parental HCC827 NSCLC (EGFR ex19 del, EGFR amp, no MET amp) by increasing concentration of erlotinib in vitro over 7 months. Model 2: ER cell line HCC827-A8 was derived from HCC827 parental xenograft tumor serially passed in vivo with long term treatment of gefitinib and erlotinib. HCC827-A8 cells express high focal MET amp, high pMET/AXL (Western blot) while retaining EGFR ex19 del (no T790M). Model 3: LU0858 was an ER patient-derived NSCLC xenograft tumor, with focal MET amp, EGFR L858R (no T790M). MET amp and EGFRmt was determined by FISH and LNA-PCR sequencing respectively. Compound dosing: emibetuzumab 20 mg/kg qw; necitumumab 4 mg/kg or 20 mg/kg biw; cetuximab 4 mg/kg biw; erlotinib 25 mg/kg qd; 5 mg/kg AZD9291 qd; 30 mg/kg CO1686 bid. Results: EGFR inhibitors, but not emibetuzumab showed significant single agent anti-tumor effect in xenograft tumors derived from non-MET amp HCC827 parental cells. In MET amp ER models, single agent emibetuzumab resulted in tumor growth inhibition in Model 1 (T/C= 51.7%-61.0%, p Conclusion: The three erlotinib resistant models with MET amp and retaining sensitizing EGFRmt (ex19 del or L858R), and no acquired T790M were found resistant to other EGFR inhibitors (Abs and TKIs). Emibetuzumab in combination with either EGFR TKI or Ab showed anti-tumor activity in MET amp ER xenograft models including tumor regression in 2 out of 3 models. The combination of emibetuzumab with erlotinib is being evaluated in NSCLC patients with EGFR activating mutation (NCT01897480). Citation Format: Suzane L. Um, Victoria L. Peek, Jennifer R. Stephens, Jessica A. Baker, Holly K. Cannon, Joel D. Cook, Isabella H. Wulur, Roger Agyei, Sudhakar Chintharlapalli, Robert J. Evans, William J. Feaver, Lysiane Huber, Linda N. Lee, Ling Liu, Liandong Ma, Ruslan Novosiadly, Volker Wacheck, Sau-Chi Betty Yan. Antitumor activity of MET antibody emibetuzumab (LY2875358) in combination with EGFR inhibitors in erlotinib resistant (ER) xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 519. doi:10.1158/1538-7445.AM2017-519

Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease with a strong association between disease and the HLA class II region. Because abnormal antigen processing, in part characterized by altered class I processing, has been identified in patients with IDDM, the TAP (transporter associated with antigen processing) genes located in the HLA class II region make attractive candidate genes for IDDM. Five coding region variants of TAP1 were typed in a cohort of well characterized Finnish patients with diabetes (n = 119) and compared to racially marched control subjects (n = 92). We found that although no single TAP1 polymorphism was associated with IDDM, a genotypic combination of Ile/Val at codon 333 with Asp/Asp at codon 637 was found more frequently in subjects with IDDM (9.4%) compared to controls (1.2%; p = 0.025). This could not be accounted for by an association with any particular haplotype defined by class I or class II serology.

Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC50=20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC50=1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED50=12.7 mg/kg) and significantly reduced (P

Recent studies suggest that multidrug resistance of HL60/ADR cells is related to an overexpression of the MRP (multidrug resistance associated protein) gene which encodes a 190-kDa ATP-binding membrane glycoprotein. In the present study we have further characterized P190 and have examined phosphorylation properties of the protein. The results demonstrate that P190 is highly phosphorylated and that the phosphate groups are metabolically active and undergo cycles of phosphorylation and dephosphorylation in the cell. Serine is the single amino acid phosphorylated in P190 and the phosphate groups are contained in nine tryptic peptides. Experiments have also been conducted to analyze the effect of various protein kinase inhibitors on phosphorylation levels of P190. The results show that H-7, staurosporine, and chelerythrine can reduce the phosphorylation of this protein. In the presence of both H-7 (200 microM) and staurosporine (200 nM) the phosphorylation of P190 is completely blocked. It has also been found that in the presence of these agents there is a major increase in drug accumulation and concomitant inhibition in drug efflux of resistant cells. These results therefore suggest the possibility that certain phosphate groups of protein P190 play an important role in modulating drug accumulation in resistant cells.

LY2457546 is a potent and orally bioavailable inhibitor of multiple receptor tyrosine kinases involved in angiogenic and tumorigenic signalling. In biochemical and cellular assays, LY2457546 demonstrates potent activity against targets that include VEGFR2 (KDR), PDGFRβ, FLT-3, Tie-2 and members of the Eph family of receptors. With activities against both Tie2 and Eph receptors, LY2457546 possesses an activity profile that distinguishes it from multikinase inhibitors. When compared head to head with sunitinib, LY2457546 was more potent for inhibition of endothelial tube formation in an in vitro angiogenesis co-culture model with an intermittent treatment design. In vivo, LY2457546 inhibited VEGF-driven autophosphorylation of lung KDR in the mouse and rat in a dose and concentration dependent manner. LY2457546 was well tolerated and exhibited efficacy in a 13762 syngeneic rat mammary tumor model in both once and twice daily continuous dosing schedules and in mouse human tumor xenograft models of lung, colon, and prostate origin. Additionally, LY2457546 caused complete regression of well-established tumors in an acute myelogenous leukemia (AML) FLT3-ITD mutant xenograft tumor model. The observed efficacy that was displayed by LY2457546 in the AML FLT3-ITD mutant tumor model was superior to sunitinib when both were evaluated using equivalent doses normalized to in vivo inhibition of pKDR in mouse lung. LY2457546 was well tolerated in non-clinical toxicology studies conducted in rats and dogs. The majority of the toxicities observed were similar to those observed with other multi-targeted anti-angiogenic kinase inhibitors (MAKs) and included bone marrow hypocellularity, hair and skin depigmentation, cartilage dysplasia and lymphoid organ degeneration and necrosis. Thus, the unique spectrum of target activity, potent in vivo anti-tumor efficacy in a variety of rodent and human solid tumor models, exquisite potency against a clinically relevant model of AML, and non-clinical safety profile justify the advancement of LY2457546 into clinical testing.

Human multidrug resistance protein 1 (MRP1) confers resistance to the Vinca alkaloids, the anthracyclines, and the epipodophyllotoxins. It is also capable of binding to and transporting the glutathione S-conjugate leukotriene C4 (LTC4) in isolated membrane vesicles. To explore species differences that exist between MRP orthologs, we cloned and characterized the mRNA encoding a canine ortholog of human MRP1-designated canine MRP1 (canMRP1). The canMRP1 mRNA encodes a protein of identical length as MRP1. Sequence alignment revealed that canMRP1 was 92% identical to MRP1 and 88% identical to murine mrp1. Five polymorphisms were identified in the canMRP1 cDNA coding sequence, including one resulting in an amino acid change from alanine to serine at aa149 (canMRP1-A and B alleles, respectively). canMRP1 was expressed and functionally characterized in HeLa and A2780 cells. Both alleles conferred an increased resistance to vincristine and etoposide and transported LTC4. The compound LY402913, a modulating agent developed against human MRP1, was able to sensitize canMRP1-expressing cells to vincristine. The modulation of canMRP1 by LY402913 was additionally confirmed by the calcein-AM accumulation assay. LY402913 inhibited the efflux of calcein in canMRP1-expressing cells. Thus, canMRP1 is similar to MRP1 in conferring resistance to vincristine and etoposide, transporting calcein-a.m., and being inhibited by LY402913. However, despite the high degree of sequence identity and functional similarity to MRP1, canMRP1 transgene failed to confer resistance to doxorubicin either in HeLa or A2780 cells. Knowledge of species differences between canine and human proteins will aid in the design of appropriate pharmacokinetic and toxicokinetic studies for the preclinical evaluation of MRP1 modulators.

Abstract 5054 Introduction: The discovery of JAK2 V617F mutation in patients with myeloproliferative disorders (MPD) has opened new perspectives for the development of targeted therapies. We have studied the efficacy of a novel molecule LY2784544 with JAK2 inhibitory activity in the in vitro growth of myeloid progenitors from JAK2 V617F-positive polycythemia vera (PV) patients. Objectives: To investigate the efficacy of LY2784544 in the inhibition of endogenous(e)BFU-E and CFU-GM growth in PV patients. Methods: In vitro cultures in semisolid media were performed from peripheral blood mononuclear cells (PBMC) of 6 PV patients who had never received cytoreductive treatment (4 patients with homozygous JAK2 V617F and 2 patients with heterozygous JAK2 V617F). PBMC were suspended in methylcellulose (Methocult. StemCell, Vancouver, Canada) without the addition of EPO and containing 0–30.0 μM LY2784544 drug. Concurrent plates containing EPO were plated as control cultures. The medium was distributed in multidishes and they were incubated at 37° with 5% CO2 and 95% humidity. Hemoglobinized colonies and granulomonocytic colonies were counted on day 14 by standard criteria (BFU-E defined by an aggregate of >50 hemoglobinized cells or three or more erythroid subcolonies and CFU-GM was defined by an aggregate of >50 cells). Each in vitro assay was performed in duplicate. DNA was obtained from peripheral blood granulocytes from each patient to quantify the JAK2 V617F allele burden at the time of culture assay. Results: LY2784544, at concentrations ranging from 0.03–30.0 μM, inhibited growth of unselected peripheral blood eBFU-E and CFU-GM from PV patients carrying the JAK2 V617F mutation in a dose-dependent manner, although without achieving complete inhibition of all colonies (fig.1). Conclusions: In vitro studies show that LY2784544 decreases the eBFU-E and CFU-GM growth in therapy-naive JAK2 V617F positive PV patients. Our data suggest that LY2784544 may be a candidate for the treatment of MPD carrying the JAK2 V617F mutation. Disclosures: Ma: Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment.

Abstract 4087 Introduction: The JAK2-V617F mutation is the most common molecular abnormality in the BCR/ABL-negative myeloid proliferative neoplasms (MPNs) and is present in approximately 95% of patients with polycythemia vera (PV) and roughly 60% of patients with either essential thrombocythemia (ET) or primary myelofibrosis (PMF). The JAK2 V617F kinase is constitutively active, oncogenic and recapitulates MPN in murine models. Therefore, mutant JAK2 is a potential therapeutic target for MPNs. Here, we describe the efficacy of the clinical candidate LY2784544; a novel small molecule inhibitor selective for JAK2 V617F mutant, in JAK2 V617F-induced in vitro and in vivo models. Methods: Assessment of the ability of LY2784544 to inhibit the signaling of the V617F mutant or wild type JAK2 was evaluated in Ba/F3 cells expressing V617F or wild type JAK2, whereby the cellular level of phospho-STAT5 (pSTAT5) was measured by a high content imaging (Cellomics) assay. The antiproliferative activity of LY2784544 and its ability to induce apoptosis were examined by Celltiter-Glo Cell Viability and Caspase-Glo 3/7 Apoptosis Assays, respectively. The effect of LY2784544 on JAK3 was investigated in IL-2 stimulated NK-92 (nature killer) cells by measuring the inhibition of JAK3-STAT5 signaling with the Cellomics assay. To examine the in vivo target inhibition, a JAK2 V617F-induced ascites tumor model was established by implanting Ba/F3-JAK2 V617F-GFP cells into the intraperitoneal cavity of severe combined immunodeficiency (SCID) mice. After orally administering LY2784544, the inhibition of pSTAT5 was measured in Ba/F3-JAK2 V617F-GFP ascites tumor cells by fluorescence activated cell sorting (FACS) assay. The anti-tumor efficacy of LY2784544 was investigated in a SCID mouse model of MPN developed by intravenously implanting Ba/F3-JAK2 V617F-GFP cells. After oral treatment for 7 or 14 days, twice daily (BID), tumor burden reduction and the effect on erythroid progenitor cells were determined by measuring the GFP positive tumor cells and CD71/Ter119 positive cells in the spleens with FACS assays, respectively. Results: In the in vitro tests using Ba/F3 cells expressing either wild type or V617F mutant JAK2, LY2784544 potently inhibited the JAK2 V617F-STAT5 signaling at a concentration that was 41-fold lower than that required to inhibit IL-3-activated wild type JAK2-STAT5 signaling (IC50=0.055 μ M for JAK2 V617F vs. 2.26 μ M for WT JAK2). Similarly in the proliferation assay, LY2784544 selectively inhibited the JAK2 V617F-driven cell proliferation (IC50= 0.068 μ M). Inhibition of JAK2 V617F signaling correlated well with the induction of apoptosis (EC50= 0.113 μ M) in Ba/F3 cell model. Consistent with the observed apparent selectivity for the V617F mutant and reduced sensitivity against wild type JAK2, a lower potency was observed with LY2784544 in the IL-3-dependent cell proliferation assay (IC50=1.356 μ M) as well as less effective inhibition of IL-2 dependent JAK3-STAT5 signaling in NK-92 cells (IC50=0.94 μ M). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2 V617F-GFP ascites tumor cells with a Threshold Effective Dose 50 (TED50) of 12.7 mg/kg. Consistent with this effective inhibition of JAK2V617F, LY2784544 also significantly reduced Ba/F3-JAK2 V617F-GFP tumor burden in the JAK2 V617F-induced MPN model with a TED50 of 13.7 mg/kg after oral treatment (BID) for 14 days, indicating that the efficacy of LY2784544 is mediated by pharmacological inhibition of JAK2 V16F-STAT5 pathway. Furthermore, LY2784544 selectively reduced Ba/F3-JAK2 V617F-GFP tumor cells with no effect on CD71/Ter119 positive erythroid progenitors in spleens of SCID mice after oral treatment (BID) for seven days. Conclusions: The in vitro data suggest that constitutively active JAK2 V617F is more sensitive to LY2784544 than cytokine-activated wild type JAK2. Consistent with this observation, in MPN disease model testing, LY2784544 selectively reduces JAK2 V617F tumor cell burden with no effect on erythroid progenitor cells. These findings suggest that as a JAK2 V617F selective targeted therapy, LY2784544 has the potential to induce apoptosis in JAK2 V617F malignant clones while potentially minimizing unintended effects on the normal hematopoietic progenitor cells. These results helped to support the advancement of LY2784544 into an ongoing phase I trial in the MPNs (I3X-MC-JHTA, NCT01134120). Disclosures: Ma: Eli Lilly and Company: Employment. Zhao:Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment. Clayton:Eli Lilly and Company: Employment. Blosser:Eli Lilly and Company: Employment. Burkholder:Eli Lilly and Company: Employment. Smith:Eli Lilly and Company: Employment.

Previous studies have suggested that vacuolar H(+)-ATPase activity may play a role in modulating drug transport mechanism in multidrug resistant HL60 cells. In the present study we have used a cDNA of human vacuolar H(+)-ATPase subunit C (SC-H(+)-ATPase) to analyze expression of this gene in HL60 cells isolated for resistance to adriamycin or vincristine. The results demonstrate that development of resistance to either agent results in a major increase in the levels of SC-H(+)-ATPase mRNA. Furthermore in resistant cells which have partially reverted to drug sensitivity there is a parallel reduction in SC-H(+)-ATPase mRNA levels. Southern blot analysis shows that the SC-H(+)-ATPase gene is not amplified in the resistant cells. These results therefore demonstrate a correlation between the development of multidrug resistance and enhanced expression of the SC-H(+)-ATPase gene.