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Breast cancer (BC) has recently surpassed lung cancer to become the most commonly diagnosed cancer in women worldwide. Approximately 70% of BC was ER+, in which estrogen receptor α (ERα, encoded by ESR1 gene) drives dysregulated cell proliferation. In clinic, ER+ BC has been effectively treated by endocrine therapy targeting estrogen or ER. As the first approved ERα degrader, fulvestrant has proven to be effective for locally advanced or metastatic BC. However, its clinical utilization is hampered by inconvenience of intramuscular injection, as well as its poor PK/PD profile and limited efficacy, especially in the patient population that developed ESR1 mutation-conferring drug resistance after the earlier line of treatment with an aromatase inhibitor. Thus, developing an oral and more effective therapy for ER+ BC remains an unmet medical need. We report herein discovery and preclinical investigation of a novel orally bioavailable SERD TFX06. TFX06 demonstrated potent binding affinity to ERα with a Ki of 0.10 nM and preferably antagonized ERα to ERβ with an IC50 value of 0.086 nM and 2.83 nM, respectively. TFX06 potently induced ERα degradation in ER+ BC cells and substantially inhibited cell proliferation in BC cell lines with ER wild type (WT), and MCF-7 cells expressing Y537S or D538G mutants. Oral administration of TFX06 demonstrated impressive antitumor efficacy in ER WT (TGI = 101%) and ER D538G-expressing (TGI = 99%) MCF-7 cell line-derived xenograft (CDX) models in mice. Similarly, in an ER+ BC patient-derived xenograft (PDX) model (HCI-013) with ER Y537S mutation, TFX06 also exerted excellent antitumor activity (TGI = 99%). Furthermore, TFX06 in combination with palbociclib achieved synergistic activity in MCF-7 CDX model. Moreover, TFX06 exhibited an excellent correlation between systemic/local drug exposure, pharmacodynamic modulation (i.e., ERα downregulation) and antitumor activity. Collectively, these findings demonstrate that TFX06 is a novel, orally bioavailable SERD. TFX06 demonstrates substantial antitumor activity in both in vitro and in vivo preclinical tumor models, including those expressing ESR1 mutations, through downregulation of ERα. The preclinical data warranted the clinical evaluation of TFX06 in human with an IND application submitted to the China NPMA. Citation Format: Jinping Wu, Ke An, Douglas D. Fang, Jianyu Lu, Lihong Hu, Jing Wang, Kaili Zhang, Yuanfeng Xia, Charles Ding, Shuhui Chen, Sha Wei. Preclinical development of an oral selective estrogen receptor degrader (SERD) TFX06 for the treatment of ER+HER2− breast cancer [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 474.

The KRAS gene, which encodes a guanosine triphosphate (GTPase) protein, is frequently mutated in solid cancers. The KRASG12C mutation aberrantly attenuates GTPase activity, leading to accumulation of the GTP-bound activated form of the KRAS protein and activation of downstream signaling pathways. This genotype occurs in approximately 13% of non-small-cell lung cancers (NSCLCs) and 1% to 3% of colorectal cancers and other solid tumors. In this study, investigational agent APG-1842 was characterized as a potent, selective, and covalent KRASG12C inhibitor that irreversibly and specifically blocks KRASG12C in an inactive guanosine diphosphate (GDP)-bound state and downregulates KRAS-dependent signaling. A RAS-GTP pull-down assay demonstrated that APG-1842 (at a concentration as low as 12 nM) completely blocked GDP-GTP nucleotide exchange, which stabilized an inactive GDP-bound form of KRASG12C and thereby inhibited RAS and RAF binding in KRASG12C-mutant human NSCLC NCI-H358 cells. In these cells, APG-1842 dose dependently inhibited KRAS-dependent signaling target proteins, including phosphorylated extracellular signal-regulated kinase (ERK), S6, and serine/threonine protein kinase Akt (protein kinase B), with a half-maximal inhibitory concentration (IC50) value of 4 nM for ERK1/2 phosphorylation. Using a panel of 18 cancer cell lines representing multiple cancer types in cell viability assays, we found that APG-1842 selectively inhibited cell proliferation in KRASG12C-mutated, but not non-KRASG12C or KRAS wild-type, cell lines. Oral APG-1842 in mice carrying cell-derived NCI-H358 NSCLC subcutaneous xenografts exerted significant antitumor efficacy at doses ranging from 3 to 100 mg/kg, resulting in tumor regression at 30 mg/kg and above. Consistent with the cellular results, APG-1842 dose dependently inhibited ERK1/2 and S6 phosphorylation in xenografts after 6 and 24 hours of treatment, correlating with systemic and tumor drug exposures. Significant antitumor activity of APG-1842 was also confirmed in a panel of 8 KRASG12C-mutated patient-derived xenograft models derived from patient samples of lung, colorectal, and gastric cancers. Collectively, these preclinical results suggest that APG-1842 is a potent, bioavailable, and highly selective KRASG12C inhibitor, laying a foundation for clinical development of this agent for patients with KRASG12C-mutant solid tumors. Citation Format: Shaoulai Gu, Qiang Li, Chao Li, Qixin Wang, Douglas D. Fang, Shaomeng Wang, Dajun Yang, Yifan Zhai. Development of covalent KRASG12C inhibitor APG-1842 for the treatment of solid tumors [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 2664.

Three core components constitute the polycomb repressive complex 2 (PRC2), a multiprotein complex that catalyzes methylation of histone H3 at lysine 27 (H3K27): (1) enhancer of zeste homolog 2 (EZH2), (2) EED, and (3) suppressor of zeste 12 protein homolog (SUZ12). Dysregulated function of PRC2 is implicated in the development of various cancer types. With regulatory approval of EZH2 inhibitor tazemetostat, a potentially effective successful cancer therapeutic strategy is now available for patients with epitheloid sarcoma and relapsed or refractory follicular lymphoma. However, EZH2 inhibitor activity might be reduced because of acquired resistance through secondary mutations in EZH2 or its inability to inhibit paralogs of EZH2 (including EZH1). Because binding of EED with trimethylated H3K27 (H3K27me3) is required to activate methyltransferase activity of EZH2, allosterically targeting EED is emerging as a novel approach to inhibit PRC2 (Qi et al, Nat Chem Biol 2017). APG-5918/EEDi-5273 is a well-documented, investigational, novel, bioactive, and potent EED inhibitor (Rej et al, JMC 2021). The aim of this study was to further characterize the anticancer therapeutic activity of APG-5918 in the preclinical setting. APG-5918/EEDi-5273 showed high-affinity binding to EED protein in a biochemical assay (IC50 = 1.2 nM). It also exerted potent antiproliferative activity in several EZH2 mutant (EZH2mut) diffuse large B-cell lymphoma (DLBCL) cell lines, with IC50 values in the nanomolar range and inhibitory effects that were approximately 5 times lower than those with tazemetostat. In a mouse xenograft model derived from EZH2mut KARPAS-422 DLBCL cells, single-agent APG-5918 conferred potent and dose-dependent antitumor activity, resulting in durable complete tumor regression that correlated with inhibition of H3K27me3, induction of PRC2 target genes, and drug exposure in tumors. In summary, our results provide scientific rationale for the clinical development of APG-5918/EEDi-5273 in EZH2mut lymphomas and, potentially, other hematologic malignancies and solid tumors. Citation Format: Saijie Zhu, Douglas D. Fang, Qiang Li, Dongmei Yang, Shoulai Gu, Shaomeng Wang, Dajun Yang, Yifan Zhai. Preclinical development of embryonic ectoderm development (EED) inhibitor APG-5918/EEDi-5273 for cancer therapy [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 3939.

The BCL-2 inhibitor venetoclax has shown impressive efficacy in patients with chronic lymphocytic leukemia, but its clinical benefits are limited by acquired BCL-2 gene mutations that confer drug resistance. Among acquired mutations, those proximal to BH3 binding motifs (e.g., G101V, D103E, and V156D) have the most significant impact on BCL-2 binding to BH3-only prodeath proteins and BH3 mimetics (e.g., venetoclax). Hence, it is important to identify novel therapeutics that address this emerging unmet need. To this end, we investigated the effect of co-targeting BCL-2 and MDM2-p53 apoptosis pathways in preclinical models of acute lymphoblastic leukemia (ALL) with BCL-2 mutations. We first genetically engineered cell lines that express clinically relevant BCL-2 gene mutations in ALL cell line RS4;11, which shows greater expression of mutants, compared to endogenous wild-type BCL-2 protein. Relative to parental cells, sensitivity to venetoclax and investigational, clinical-stage BCL-2 inhibitor lisaftoclax (APG-2575) in mutant cell lines was reduced 160-fold. Using these BCL-2-inhibitor-resistant cell lines, we assessed the effect of combining lisaftoclax with investigational, clinical-stage MDM2-p53 inhibitor alrizomadlin (APG-115). Alrizomadlin inhibits the MDM2-p53 interaction and activates the prodeath p53 tumor suppressor signaling pathways (Aguilar A et al. J Med Chem 2017). Also potentially consequential was the observation that combining alrizomadlin with lisaftoclax synergistically inhibited proliferation and induced apoptosis in RS4:11 cells carrying G101V, V156D, or D103E mutations. Treatment with this combination also synergistically enhanced antitumor activity in mouse xenograft tumor models derived from RS4:11 cells carrying G101V or V156D mutations. In these animal models, lisaftoclax alone exhibited no or limited activity, with T/C (%; i.e., relative tumor volume of treatment group vs. control) values ranging from 86.40% to 99.15%. In contrast, single-agent alrizomadlin exerted slightly more potent activity, with T/C values ranging from 57.25% to 62.35%. Importantly, the combination of lisaftoclax and alrizomadlin demonstrated synergy, with T/C values ranging from 12.30% to 32.16%. Mechanistically, alrizomadlin likely induced expression of prodeath BCL-2 family proteins BAX, PUMA, and Noxa, which are p53 target genes. Further, lisaftoclax plus alrizomadlin more effectively blocked cell cycle entry into the G2/M phase, resulting in accumulation of sub-G1 apoptotic cells. In summary, our study demonstrates, for the first time, that co-targeting BCL-2 and MDM2-p53 apoptosis pathways overcame resistance to BCL-2 inhibitors conferred by acquired BCL-2 gene mutations, potentially offering a viable strategy to overcome drug resistance and thus providing a rationale for clinical investigation. Citation Format: Jing Deng, Kaixiang Zhang, Qiuqiong Tang, Xiaojing Huang, Qixin Wang, Na Li, Douglas D. Fang, Dajun Yang, Yifan Zhai. Co-targeting MDM2-p53 and BCL-2 apoptosis pathways overcomes resistance conferred by acquired BCL-2 gene mutations in preclinical models [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 3964.

Mouse double minute-2 (MDM2)-p53 inhibitor alrizomadlin (APG-115) is an investigational agent known to induce apoptosis of TP53-wild type cancer cells (Aguilar et al, J Med Chem 2017). Emerging evidence suggests that activation of p53 by alrizomadlin also promotes antitumor immunity in the tumor microenvironment (Fang et al, JITC 2019; Zhou et al, Nat Immunol 2021), but the links between these processes are incompletely understood. Pyroptosis refers to inflammatory programmed cell death. Central to this process is the family of gasdermins, which can form pores in cell plasma membranes, resulting in lysis and release of immune stimulants. In cells expressing these proteins, GSDME can be cleaved by caspase-3, which converts noninflammatory apoptosis to pyroptosis (Zhang et al, Nature 2020). In this context, caspase-3/GSDME appears to represent a switch between apoptosis and pyroptosis. Given that alrizomadlin elicits its apoptogenic activity primarily by activating caspase-3, we hypothesized that the MDM2-p53 inhibitor might also induce pyroptosis in GSDME-expressing cells by cleaving caspases. Our study demonstrated that alrizomadlin uniformly elicited robust pyroptosis in GSDME-expressing cancer cells. After treatment with alrizomadlin, the dying cells showed swelling with plasma membrane blebbing characteristic of pyroptosis. Alrizomadlin treatment upregulated biomarkers of pyroptosis, inducing activation of caspase-3, promoting cleavage of GSDME, and increasing release of lactate dehydrogenase (LDH). After a short drug exposure of 24 hours, alrizomadlin elicited concurrent apoptotic and pyroptotic tumor cell death, as evidenced by coexistence of the hallmarks of apoptosis (PARP-1 cleavage) and pyroptosis (caspase-3 cleavage and activated GSDME) markers. When the treatment duration was extended to 48 hours, cleavage of GSDME continued to increase whereas PARP-1 cleavage became nearly undetectable, suggesting a conversion from apoptosis to pyroptosis. Inhibition of caspases by pan-caspase inhibitor Q-VD-OPh (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone) abolished not only alrizomadlin-induced caspase-3 and GSDME cleavage but also reduced LDH release and cell death without inducing morphologic changes associated with pyroptosis. Taken together, these results suggest that, in addition to apoptosis, MDM2-p53 inhibitor alrizomadlin induces caspase-mediated pyroptosis in GSDME-expressing cancer cells. In this study, we reveal for the first time that apoptosis-inducing, alrizomadlin induces both apoptosis and pyroptosis in GSDME-expressing cancer cells. GSDME-dependent pyroptosis may be a previously unrecognized mechanism of action for alrizomadlin to exert antitumor immunity, with potentially important implications for clinical development of therapy involving MDM2-p53 inhibition. Citation Format: Qiuqiong Tang, Douglas D. Fang, Huidan Yu, Bingxing Wu, Yan Yin, Dajun Yang, Yifan Zhai. Inhibition of MDM2-p53 interaction by alrizomadlin (APG-115) induces pyroptotic cell death in gasdermin E (GSDME)-expressing cancer cells [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 2998.

The AML treatment landscape has improved dramatically in the past decade, with improved objective response rates and overall survival after treatment with newly approved targeted therapies. BCL-2 inhibition combined with a hypomethylating agent or low-dose cytosine arabinoside (Ara-C) is also effective in previously untreated elderly patients who are not candidates for standard induction therapy. However, the effect of BCL-2 inhibition is commonly weakened when tumor cells upregulate antiapoptotic protein MCL-1 to escape apoptosis. Thus, combination therapy is introduced to suppress MCL-1 levels. HHT (omacetaxine mepesuccinate) has been widely used in Chinese patients with AML for 30 years. As an inhibitor of protein synthesis, HHT decreases MCL-1. This study investigated the effect of combining clinical stage BCL-2 selective inhibitor APG-2575 with HHT in AML and MDS cells, as well as murine xenograft tumor models. In AML (MV-4-11, OCI-AML-3) and MDS (SKM-1) cell lines, single-agent HHT exerted stronger antiproliferative and apoptogenic activities compared to APG-2575 (as assessed by Cell Titer Glo assay or flow cytometry). When combined, these treatments synergistically inhibited cellular proliferation and induced apoptosis. In a mouse xenograft tumor model derived from MV-4-11 cells, APG-2575 demonstrated limited antitumor activity, with a T/C (Treated/Control tumor volume %) value of 63% whereas HHT showed stronger activity, with T/C equal to 30%. Most importantly, these antitumor effects were potentiated when APG-2575 and HHT were combined, yielding a T/C value of 3% and partial regression in all treated tumors. Mechanistically, BCL-2 inhibition with APG-2575 decreased BCL-2:BIM complexes and but increased MCL-1:BIM complexes, as liberated BIM was re-sequestered by MCL-1. On the other hand, concomitant treatment with HHT abolished induction of MCL-1:BIM complexes. Besides BIM complexes, MCL-1:PUMA and MCL-1:BAK pairings also decreased after exposure to HHT. The decrease in MCL-1 complex coincided with its downregulation at protein levels. Thus, under such an MCL-1-supressed condition facilitated by HHT treatment, the freed proapoptotic proteins (BIM, PUMA, BAK) more potently manifested apoptotic signals triggered by APG-2575. In summary, APG-2575 synergizes with HHT to potentiate antitumor activity in preclinical models of AML/MDS. HHT suppresses MCL-1 protein, preventing or abolishing formation of MCL-1:BIM, MCL-1:PUMA, and MCL-1:BAK complexes, and hence allowing prodeath proteins to fully engage in tumor cell apoptosis induction. Our results provide scientific rationale for clinical development of APG-2575 plus HHT in patients with AML/MDS. Citation Format: Douglas D. Fang, Qiuqiong Tang, Yanhui Kong, Tao Rong, Qixin Wang, Jing Deng, Dajun Yang, Yifan Zhai. BCL-2 inhibitor APG-2575 and homoharringtonine (HHT) synergistically induces apoptosis and inhibits tumor growth in preclinical models of acute myeloid leukemia and myelodysplastic syndromes (AML/MDS) [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 981.

Current targeted therapies such as VEGFR inhibitor sunitinib and mTOR inhibitor everolimus extend progression-free survival (PFS) in patients with Grade 1 (G1) and G2 neuroendocrine tumors (NET), but the objective response rate remains low. The median PFS in patients with advanced neuroendocrine carcinoma (NEC) receiving platinum-based therapy is only 3 to 4 months. Hence, more effective therapy is needed to improve clinical outcomes. This study explored if BCL-2 family antideath proteins play a role in NEN tumorigenesis and whether clinical-stage dual BCL-2/BCL-xL inhibitor APG-1252 might overcome intrinsic apoptotic blockade in NEN. A panel of six NEN cell lines were exposed to APG-1252-M1, which is an active metabolite of APG-1252 typically used for in vitro studies. After 72 hours, the IC50 of APG-1252-M1 ranged from 0.43 to 10 μM. Among the panel, pancreatic NET (pNET) cell lines BON-1 and β-TC3 exhibited the highest sensitivity, with IC50 values of 0.43 and 0.55 µM, respectively, whereas NEC cell line NCI-H460 was insensitive to APG-1252-M1. Interestingly, Western blot analysis revealed higher expression of BCL-xL compared to BCL-2 and MCL-1 proteins in NET cell lines (BON-1, QGP-1, NCI-H727). Higher baseline levels of BCL-xL:BIM and BCL-xL:PUMA complexes in NEN cells indicated APG-1252-M1 sensitivity. Exposure to APG-1252-M1 disrupted these complexes, liberating BIM/PUMA to trigger the apoptotic cascade, as indicated by an increase in BAX:BAK complexes. Conversely, lower BCL-xL complex levels and higher MCL-1 levels correlated with insensitivity of NCI-H460 to APG-1252-M1. Immunohistochemistry staining of BCL-xL, BCL-2, and MCL-1 proteins was performed in formalin-fixed paraffin-embedded tissue sections of 106 NEN patient tumors (including stomach/intestine/pancreatic NEN), of which approximately 70% were NET and 30% NEC. Similar to data in NEN cell lines, BCL-xL expression was higher than BCL-2 expression in NEN patient samples, in which BCL-xL expression was indicated by more than 80% of samples with IHC scoring ≥6 on a scale of 0 to 10, and BCL-2 expression by 30% IHC scoring < 6 or undetectable (60% IHC scoring 0). Unexpectedly, however, MCL-1 expression was also high, with more than 80% of samples IHC scoring ≥6. In summary, the results suggest that BCL-xL plays an important role in NEN. Cellular sensitivity to BCL-2/BCL-xL inhibitor APG-1252-M1 correlates with baseline BCL-xL complex levels. In NEN patient samples, MCL-1 was also highly expressed, implicating its potential negative regulatory effect on sensitivity to APG-1252. Concurrent expression of BCL-xL and MCL-1 proteins suggests that a combination treatment targeting both proteins might be more effective in NEN. Our findings inform development of a BCL-2/BCL-xL inhibitor for NEN therapy. Citation Format: Yu Guo, Jing Deng, Lin Zhang, Xiaojing Huang, Kaixiang Zhang, Feng Zhou, Yuanbao Li, Fan Luo, Douglas D. Fang, Dajun Yang, Jie Chen, Yifan Zhai. Targeting BCL-xL addiction with APG-1252 (pelcitoclax) to overcome apoptotic blockade in neuroendocrine neoplasm (NEN) [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 984.

Ovarian cancer is one of the deadliest malignancies in women, and up to 70% of patients with epithelial ovarian cancer have FAK overexpression, amplification, or activation. FAK plays an important role in cellular migration, growth factor signaling, cell cycle progression, cellular survival and chemoresistance. This biomarker is also significantly associated with higher tumor stage, metastasis, and shorter overall survival in patients with ovarian cancer. Inhibition of FAK is therefore emerging as a promising treatment target. APG-2449 is a clinical stage FAK/ALK/ROS1 multi-kinase inhibitor. In this study we investigated antitumor activity of APG-2449 combined with standard-of-care chemotherapeutics in ovarian cancer in the preclinical setting. In a mouse xenograft tumor model derived from ovarian cancer cell line OVCAR-3, which was resistant to platinum-based therapies, APG-2449 combined with paclitaxel, and paclitaxel plus carboplatin, synergistically enhanced antitumor activity, whereas the chemotherapeutics showed no activity. Synergistic antitumor activity was also observed in multiple patient-derived xenograft (PDX) models derived from women with chemoinsensitive ovarian cancer, which also frequently expresses high levels of FAK. By comparing gene expression profiles of PDX tumors obtained from responders and nonresponders to the combined therapy, we identified CD44 (a marker for cancer stem cells) as a potentially predictive biomarker. Western blot analysis confirmed higher protein levels of CD44 in pretreated tumors of responders. Interestingly, downregulation of CD44 levels was observed in combination-treated tumors, suggesting that these combinations reduced cancer stem cell populations in ovarian cancer. Accordingly, in ovarian cancer cells exposed to APG-2449 alone or combined with paclitaxel, numbers of cells positive for CD44 or aldehyde dehydrogenase 1 (ALDH1; another marker for cancer stem cells) decreased in a dose-dependent manner. In summary, our data suggest that FAK inhibition by APG-2449 sensitizes ovarian tumors to chemotherapeutics in preclinical tumor models of ovarian cancer. The synergistic antitumor activity was mediated by downregulation of CD44+ or ALDH1+ cancer stem cell populations. These findings encourage clinical development of APG-2449 in combination with chemotherapies for treatment of ovarian cancer. Citation Format: Ran Tao, Douglas D. Fang, Yuanbao Li, Kaixiang Zhang, Chunhua Xu, Xu Fang, Qixin Wang, Dajun Yang, Yifan Zhai. Focal adhesion kinase (FAK) inhibitor APG-2449 sensitizes ovarian tumors to chemotherapy via CD44 downregulation [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 968.

AML accounts for 80% of acute leukemias in adults. FLT3 gene mutations are observed in approximately 30% of patients with AML and augur a poor prognosis. Despite antitumor effects of selective FLT3 inhibitors, resistance to these agents continues to pose a formidable clinical challenge. The expression of pro-survival protein BCL-2 is frequently dysregulated, conferring resistance to FLT3 inhibitors, in AML. In this study, we explored the combination of clinical stage multikinase inhibitor HQP1351 (which also targets FLT3) and BCL-2-selective inhibitor APG-2575 in preclinical models of FLT3-mutant AML. Antileukemic activity of HQP1351 was first examined using AML cell lines MV-4-11 and MOLM-13, which harbor FLT3-internal tandem duplication (FLT3/ITD) mutations. In both cell lines, HQP1351 dose-dependently induced cellular apoptosis, APG-2575 also induced apoptosis, and their combination synergistically induced this process. In particular, treatment of subcutaneous MV-4-11 xenograft tumors in nude mice with HQP1351 10 mg/kg significantly suppressed tumor growth, with a tumor growth inhibition (TGI) of 72.4%. This antitumor effect was potentiated when APG-2575 was combined with HQP1351, resulting in a TGI of 97.2%. The combination also improved survival of tumor-bearing mice with a systemic MOLM-13 model compared to either agent alone. Mechanistically, HQP1351 likely inhibited phosphorylation of FLT3 and its downstream signaling pathways, including phosphorylation of AKT, ERK1/2 and STAT5. Taken together, these findings indicate that the antileukemic activity of olverembatinib can be ascribed to on-target inhibition. Single-agent APG-2575 agent triggered cellular apoptosis, as evidenced by cleavage of caspase-3 cleavage and poly(ADP-ribose) polymerase-1 (PARP-1) activation, which are considered hallmarks of apoptosis. Consequently, APG-2575 treatment also increased expression of another antiapoptotic protein (MCL-1), upregulation of which is a pivotal mechanisms of resistance to BCL-2 inhibitors. Notably, HQP1351 substantially decreased cellular MCL-1 expression when combined with APG-2575, further enhancing apoptosis.Taken together, our data suggest that FLT3 inhibition by HQP1351 downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 to potentiate cellular apoptosis in FLT3-mutant AML. The results provide scientific rationale for clinical development of HQP1351 combined with APG-2575 in patients with FLT3-ITD-mutant AML. Citation Format: Douglas D. Fang, Hengrui Zhu, Qiuqiong Tang, Qixin Wang, Na Li, Xu Fang, Ping Min, Guangfeng Wang, Dajun Yang, Yifan Zhai. FMS-like tyrosine kinase 3 (FLT3) inhibition by olverembatinib (HQP1351) downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 in preclinical models of FLT3-mutant acute myeloid leukemia (AML) [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 1096.

TNF-related apoptosis-inducing ligand (TRAIL) receptor (DR4 or DR5) agonists are promising agents for cancer therapy because they induce apoptosis selectively in cancer cells. However, their clinical effect is hampered by either primary or acquired resistance in cancer cells. Second mitochondria-derived activator of caspase (Smac) mimetics that antagonize the IAPs potently sensitize cancer cells to TRAIL-induced apoptosis in a caspase-8-dependent manner. We evaluated antitumor effects of small-molecule IAP antagonist APG-1387combined with an agonist mouse mAb directed against TRAIL death receptor type 5 (DR5) termed CTB-006 in the preclinical setting. Both agents are in phase I/II clinical development for patients with solid tumors. In vitro the combination treatment showed synergy in a dose-dependent manner across various cancer cell lines, as evidenced by synergistic antiproliferative activity and enhanced induction of caspase-3 cleavage. Potential synergistic in vivo antitumor activity of the combination was then assessed in human triple negative breast cancer (TNBC) xenograft models in mice. Specifically, in MDA-MB-231 and 2LMP TNBC xenograft models, either APG-1387 or CTB-006 single agent exhibited limited antitumor activity, with T/C (%) values of >35% (T, tumor volume in the treatment group; C, tumor volume in the vehicle control group). Only one tumor showed partial regression (PR) by DR5 treatment. Addition of APG-1387 potentiated this effect, with the combination treatment leading to T/C values of 3% to 15%, and complete regression, PR, or stable disease in all treated tumors. In summary, our results suggest great potential of combination therapy with APG-1387 and CTB-006 for solid tumor therapy and deserves further clinical investigation. Citation Format: Qiang Li, Douglas D. Fang, Zhe Li, Dajun Yang, Fengqi Cao, Yifan Zhai. Therapeutic potential of inhibitor of apoptosis protein (IAP) inhibitor APG-1387 combined with DR5 agonist monoclonal antibody (mAb) CTB-006 in preclinical models of solid 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 1924.

Background MCL-1 is an important anti-death BCL-2 family protein and plays a key role in blocking apoptosis in cancer cells. MCL-1 gene is located in one of the most frequently amplified locus in various hematologic malignancies and solid tumors, including prostate, lung, pancreatic, breast, ovarian and cervical cancers, as well as melanoma, B-cell chronic lymphocytic leukemia (B-CLL), acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)); moreover, MCL-1 overexpression is implicated as a resistance factor for multiple therapies including widely prescribed microtubule-targeted agents for breast cancers. Therefore, MCL-1 is an attractive therapeutic target for the treatment of cancers. In this study, we discovered the lead preclinical compound APG-3526, from its chemical synthesis optimization to potent antiproliferative and antitumor activity using multiple in vitro and in vivo xenograft models, respectively. Methods and Experiments APG-3526 was chemically synthesized by using one convergent multistep synthesis in gram scale. The binding affinity of APG-3526 was determined by a FP assay using a fluorescein labelled peptide (FAM-Bid) which binds to the MCL-1 protein leading to an increased anisotropy measured in milli-polarization (mP) values. The effect of APG-3526 on cell viability was determined using CCK-8 assay and its antitumor activities were tested in NCI-H929 and OPM-2 multiple myeloma (MM) xenograft SCID mice model (dosed i.v. at 50 mg/kg and p.o. at 25 mg/kg). Results Our biochemical study has demonstrated that APG-3526 binds to MCL-1 with a nanomolar affinity (IC50 = 7 nM). It is highly potent in inhibition of cell growth in MCL-1-dependent multiple myeloma cell lines (NCI-H929 cells: IC50 = 14 nM; OPM-2 cells: IC50 = 62 nM). Administration of APG-3526 (p.o. at 25 mg/kg, QDx14D, or i.v. at 50 mg/kg, once) achieved complete tumor regression in both NCI-929 and OPM-2 multiple myeloma xenograft models. The pharmacodynamics (PD) study using tumor samples further revealed caspase 3 activation and PARP cleavage triggered by APG-3526. MCL-1 complex MSD assay confirmed that APG-3526 disrupted MCL-1:BIM binding thus freeing BIM to initiate the apoptotic cascade. In conclusion, we have developed a novel and highly potent MCL-1 inhibitor, APG-3526, which displays clinically relevant pharmacokinetic properties and elicits potent antiproliferative and antitumor activities via disrupting MCL-1 complex and triggering caspase activation, especially in MCL-1 driven MM models. These results support APG-3526 as a promising MCL-1inhibitor for further clinical development. Citation Format: Jianyong Chen, Chengzhe Wu, Lingling Jiao, Leilei Zhao, Yunlong Zhou, Dongbo Li, Guozhi Tang, Shoulai Gu, Jing Deng, Guangfeng Wang, Douglas D. Fang, Shaomeng Wang, Dajun Yang, Yifan Zhai. Development of APG-3526 as a novel and highly efficacious MCL-1 inhibitor [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 73.

Background: Gastric cancer is one of the most prevalent cancers in the East Asia population. The five-year survival rate is approximately 20% globally. The analysis of gene expression data suggests that anti-apoptotic protein BCL-xL may be the oncogenic driver in gastric cancer as its expression levels are much higher than BCL-2 (Pan Cancer Atlas). In this study, we investigated if a clinical stage BCL-2/BCL-xL dual inhibitor APG-1252 would elicit therapeutic activity and associated mechanisms using a panel of gastric cancer patient-derived xenograft (PDX) models. Additionally, we explored if the combination with HER2 inhibition could enhance the antitumor activity in HER2+ gastric cancer models. Methods and Experiments: Eighteen PDX models of gastric cancer that bore high levels of BCL-xL expression (BCL-xLhigh) based on the gene expression profiles were selected for a mouse trial (n=2) with a dosage regimen of APG-1252 (100 mg/kg, BIW, IV) for three weeks. In a combination study (n=5), APG-1252 (50 mg/kg, BIW, IV) and lapatinib (100 mg/kg, QD, PO) were concurrently administered for three weeks. An advanced ELISA (MSD) and Western blot analysis were performed to detect BCL-xL complex and the protein level, respectively, in tumor samples. Results: APG-1252 treatment resulted in tumor growth inhibition (TGI greater than 50%) in four PDXs (20%), including three exhibiting TGI greater than 80%. PD study confirmed that BCL-xL protein levels were high, and bound with pro-apoptotic proteins PUMA (major binding partner) and BIM in the xenograft tumors of these models. Generally, in comparison with hematologic cancer cells which exhibit dominant BCL-2 complexes (i.e., Toledo), the levels of BCL-xL complexes were consistently higher than those of BCL-2 complexes in the gastric cancer PDXs, implicating a crucial role of BCL-xL in the solid tumors. After treatment with APG-1252, BCL-xL:PUMA, were disrupted and, interestingly, such a decrease in BCL-xL complex signal correlated with TGI. Additionally, the baseline levels of BCL-xL complexes also correlated with TGI. Furthermore, a consequent increase in MCL-1 complexes after APG-1252 treatment indicated that such a compensatory mechanism among anti-apoptotic proteins may confer the drug resistance. Finally, in mouse tumor models derived from HER2+ gastric cancer cell line NCI-N87 or patient-derived xenografts (PDXs), combined treatment with AGP-1252 and HER2 inhibitor lapatinib synergistically inhibited tumor growth, suggesting that lapatinib may be able to increase mitochondrial priming, thus sensitizing cancer cells to APG-1252. Conclusion: Our results demonstrate the on-target antitumor activity of APG-1252, the potential of BCL-xLhigh as a predictive biomarker, and the resistance mechanism conferred by MCL-1. Furthermore, the data provide a scientific rationale for the combined therapy with BCL-xL and HER2 inhibitors to achieve better clinical outcomes in a subset of HER2+ gastric cancers. Citation Format: Jing Deng, Xiaojing Huang, Guoqin Zhai, Kaixiang Zhang, Jiaxing Gu, Tingting Mao, Yan Yin, Ran Tao, Douglas D. Fang, Dajun Yang, Yifan Zhai. Co-targeting BCL-xL and HER2 high expression to overcome apoptosis blockade in gastric cancer [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 74.

HQP1351 is a novel, orally bioavailable multi-kinase inhibitor targeting BCR-ABL, KIT, and FLT3. Currently, HQP1351 is in phase II clinical trials in relapsed and refractory chronic myeloid leukemia (CML) patients by targeting BCR-ABL. Besides, HQP1351 inhibits both wild-type and mutant FLT3 in kinase binding assay. APG-115 is another clinical stage, small molecule MDM2 antagonist. In the present study, we explored the antitumor effect of the combination of HQP1351 and APG-115, and the molecular mechanism in FLT3-ITD and TP53 wild-type acute myeloid leukemia (AML) in the preclinical setting. First, the effect of HQP1351 as single agent on cell viability in FLT3-ITD mutant and TP53 wild-type human AML cell lines, including MV-4-11 and MOLM-13. Second, antitumor activity of the combination was investigated in systemic and subcutaneous xenograft models in NOD/SCID mice derived from these cells. The results showed that HQP1351 alone exhibited potent antiproliferative activity in both cell lines in vitro, with nanomolar IC50 values. The activity was enhanced in the combination treatment with APG-115. In vivo, HQP1351 single agent demonstrated significant antitumor activity evidenced by a markedly reduction of tumor burden (i.e., CD45+ human tumor cells) in systemic MOLM-13 xenograft model. Treatment with HQP1351 at 3, 10 and 30 mg/kg significantly prolonged mice survival with median survival of 20.5 days, 26.0 days and 35 days, respectively, compared with 18 days in the control group. In subcutaneous MV-4-11 xenograft model, treatment with HQP1351 or APG-115 single agents achieved T/C values of 28.6% and 59.6%, respectively. The combination achieved synergistic antitumor activity with a T/C value of 13.4%. The benefit of the combination was also demonstrated in systemic MOLM-13 xenograft model. Mechanistically, the combined treatment synergistically downregulated p-FLT3, p-ERK, p-STAT5 and anti-apoptotic protein MCL-1, and thus enhanced antitumor effect. Taken together, our data provide scientific rationale for clinical development of the combination of HQP1351 and APG-115 in FLT3-ITD mutant and TP53 wild-type AML patients. Citation Format: Douglas D. Fang, Qiuqiong Tang, Qixin Wang, Na Li, Xu Fang, Jiaxing Gu, Yanhui Kong, Tao Rong, Guangfeng Wang, Dajun Yang, Yifan Zhai. Synergy of tyrosine kinase inhibitor HQP1351 and MDM2-P53 antagonist, APG-115, in preclinical models of FLT3 mutant and TP53 wild-type acute myeloid leukemia [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 4217.

Osimertinib (AZD9291) is the first-line treatment for EGFR-mutated NSCLC; however, the majority of patients inevitably develop resistance due to de novo genomic abnormalities, such as C797S mutation, EGFR exon 20 insertion, MET amplification and other unknown mechanisms. Hence, effective therapies to overcome acquired resistance are urgently needed. Inhibition of BCL-2/BCL-xL has been reported to enhance apoptosis in EGFR-TKI resistant cells with low sensitivity to EGFR inhibition. In this study, we evaluated whether the combination of a dual BCL-2/BCL-xL inhibitor APG-1252 and chemotherapeutics could overcome osimertinib resistance in preclinical xenograft models. First, EGFR C797S mutation was introduced to human NCI-H1975 NSCLC cells to construct an NCI-H1975-C797S resistant cell line using CRISPR technology. NCI-H1975-C797S derived-xenografts were treated with APG-1252, cisplatin/docetaxel or their combinations. Combination therapy with APG-1252 and cisplatin or docetaxel exhibited synergistic antitumor activity. APG-1252 plus docetaxel combination achieved 100% tumor partial regression (PR). Similar results were demonstrated in a patient-derived xenograft (PDX) tumor model derived from an osimertinib-resistant NSCLC patient harboring 19del-T790M-C797S mutations. Furthermore, the combinations also exhibited enhanced antitumor activity in an osimertinib-resistant PDX model that the resistant mechanism remained unknown. In summary, our results suggest that the combination treatment with APG-1252 and chemotherapeutics can overcome acquired resistance to osimertinib and the combination deserves further clinical evaluations. Citation Format: Ran Tao, Guangfeng Wang, Douglas D. Fang, Guoqin Zhai, Yuanbao Li, Jing Lv, Miaoyi Wu, Yangfeng Ge, Feifei Zhang, Danyi Wen, Dajun Yang, Yifan Zhai. Combination of BCL-2/BCL-xL dual inhibitor APG-1252 and chemotherapeutics overcomes resistance to osimertinib in EGFR mutant NSCLC in preclinical models [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 6223.

APG-1387 is a small molecule antagonist of the inhibitor of apoptosis proteins (IAPs) which blocks the activity of IAPs family proteins (XIAP, cIAP-1, cIAP-2, and ML-IAP) and consequently induces apoptosis. It is currently in phase I clinical development in patients with solid tumors, including pancreatic cancer. Targeting IAPs has been reported to effectively inhibit tumor growth in the preclinical models of pancreatic cancer. Currently, PARP and MEK inhibitors are also in clinical development for pancreatic cancer with BRCA1/2 or KRAS mutations. To further explore the therapeutic potential of APG-1387 in combination with PARP or MEK inhibitors, a genomic biomarker guided avatar mouse trial (n=2) using pancreatic cancer patient-derived xenografts (PDX) was conducted to evaluate antitumor activity of APG-1387 in combination with (i) the PARP inhibitor, olaparib, in PDXs carrying BRCA1/2 mutations, and (ii) the MEK inhibitor, trametinib, in PDXs carrying KRAS mutations. The results revealed that APG-1387 improved the antitumor activity of olaparib in 4 out of 6 (67%) BRCA1/2 mutant PDXs with a 44% reduction of T/C (%) value (T, treatment group, C, vehicle control), including one partial tumor regression (PR). In combination with trametinib, the antitumor activity was improved in 4 out of 9 (44%) KRAS mutant PDXs, showing a 34% reduction in the T/C value. Furthermore, synergy of the combinations of APG-1387 with PARP or MEK inhibitor was confirmed in a follow-up efficacy study (n=5) using a PDX model carrying both BRCA2 and KRAS mutations. The molecular mechanisms underlying the synergy are currently being investigated. In summary, our results suggest that APG-1387 is a potential agent for pancreatic cancer treatment, ascribing to its potential synergistic antitumor effect by combining with either PARP inhibitors in BRCA1/2 mutant or MEK inhibitors in KRAS mutant pancreatic cancers. Overall, our data provide the scientific rationale for the future clinical development of these combinations in pancreatic cancer patients with distinct genomic alterations. Citation Format: Qiang Li, Douglas D. Fang, Shoulai Gu, Dajun Yang, Yifan Zhai. Therapeutic potential of IAP inhibitor APG-1387 in combination with PARP- or MEK-targeted therapy, or chemotherapy in pancreatic cancer [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 6216.

Liposarcoma is a rare but highly recurrent soft tissue neoplasm. Combination of surgery and radiotherapy is the main treatment for liposarcoma patients while chemotherapy remains experimental and limited depends on disease subtypes. Well-differentiated and dedifferentiated liposarcoma (WDLPS/DDLPS) represents ~50% of all diagnosed liposarcoma and their benefit from chemotherapy has been documented to be minimal. Concomitant amplifications of MDM2 and CDK4 are frequently found in WDLPS/DDLPS patients, suggesting that the combination with two targeted agents may worth further development for the treatment. APG-115 is a potent, selective, orally bioavailable small molecule inhibitor of MDM2-p53 protein-protein interaction and thus activates tumor suppression activity of p53 in p53 wild-type tumors. Currently, APG-115 is in clinical development as a single agent or in combination with PD-1 blockade immunotherapy for solid tumors, including sarcoma. To further explore the sensitive patient population and predictive biomarkers for APG-115, in this study, we evaluated the effect of the combined MDM2/CDK4 inhibition in p53 wild-type, MDM2 and CDK4 amplified liposarcoma using a preclinical xenograft tumor model derived from a dedifferentiated liposarcoma patient (PDX). Tumor-bearing mice were treated with MDM2 inhibitor APG-115, CDK4 inhibitors palbociclib/ribociclib, or their combinations. In the first experiment, APG-115 administered at 40 mg/kg as a single agent demonstrated antitumor activity in the DDLPS PDX model. In comparison with the single agents, the combination treatment with APG-115 and palbociclib or APG-115 and ribociclib exerted synergistic antitumor effects, resulting in T/C values of 16.9% and 27.1%, respectively (T/C value, average tumor volumes in treatment group vs. the vehicle control group). Partial tumor regression (PR) was achieved in 1/5 animals in both combination treatment groups but not in single arms. Another independent experiment with a higher dose of APG-115 (80 mg/kg) combined with palbociclib showed further improved antitumor activity with T/C value of 7.0% (2/5 PR). Collectively, our results suggest that the combination therapy of APG-115 and a CDK4 inhibitor may benefit WDLPS/DDLPS patients with p53 wild-type, and MDM2 and CDK4 amplifications clinically. Citation Format: Douglas D. Fang, Guoqin Zhai, Chunhua Xu, Qingyang Gu, Jingwen Wang, Saijie Zhu, Dajun Yang, Yifan Zhai. MDM2 inhibitor APG-115 synergizes with CDK4/6 inhibitors in a patient-derived xenograft model of dedifferentiated liposarcoma [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 1253.

Background: BCL-2, an anti-apoptotic protein and oncogene, has been considered one of the top 10 targets for cancer drug development. The inhibition of BCL-2 and other anti-apoptotic proteins will help to restore the apoptotic pathway. Aiming to target much diverse BCL-2 family protein dependency in solid tumors, we have developed a potent BCL-2 and BCL-xL dual inhibitor APG-1252, which is currently in phase 1 trials for solid tumors in USA, China and Australia. Mechanistically, BCL-2 and BCL-xL protect cells from cell death by sequestering pro-death BH3-only proteins like BIM. Thus, BCL-2 and BCL-xL inhibitors are designed to disrupt the protein complexes and release pro-death proteins to trigger downstream apoptotic cascade. In this study, we intended to further understand the mechanism of action of APG-1252 in vivo and factors regulating its sensitivity. To this end, we conducted trials in SCID mice carrying patient-derived xenograft (PDX) tumors, and studied target engagement. Methods and Experiments: We first selected eleven gastric and esophageal cancer PDX models that bore high levels of BCL-xL (BCL-xLhigh) but normal levels of MCL-1 (MCL-1nor) based on the gene expression profiles and the consideration of that BCL-xL may play a major role in tumorigenesis in solid tumors and MCL-1 a resistant factor for the dual inhibitor. Mice bearing PDX tumors were intravenously treated with 100 mg/kg of APG-1252 twice per week for three weeks. At the end of treatments, tumor samples were collected. An advanced ELISA system (MSD, Meco Scale Discovery) and Western blotting were performed to detect BCL-2 and BCL-xL protein complex and individual proteins. Results: 1. The MSD assays revealed dominant BCL-xL:BIM complex signals in the solid tumor PDX samples in comparison with hematological malignancy cell lines, in which BCL-2:BIM was the major complex. 2. The dual inhibitor treatment disrupted BCL-xL:BIM complex, thus BCL-xL:BIM complex may serves as a pharmacodynamic (PD) marker for target engagement. 3. BCL-2 family protein profiles showed high BCL-xL and MCL-1 levels in the solid tumor PDXs. The treatment with APG-1252 led to a significant increase in BCL-2 protein levels, while there was no major change on pro-apoptotic BH3-only proteins BIM and PUMA. 4. The more efficacious responders to the treatment exhibited a more substantial decrease in BCL-xL:BIM complex signals and low MCL-1 protein levels, whereas a much less significant decrease in BCL-xL:BIM complex and higher MCL-1 levels were found in the poor or non-responders. Conclusion: Both BCL-xL protein and BCL-xL:BIM complex are dominant in the solid tumor PDX tissues. The decrease of BCL-xL:BIM complex indicates the on-target activity after the treatment with APG-1252. Thus, the complex may be used to monitor the target engagement and, potentially, a predictive biomarker for APG-1252 efficacy. These assays may be applied to patient samples to guide the clinical trials. Citation Format: Jing Deng, Xiaojing Huang, Guoqin Zhai, Tingting Mao, Yan Yin, Douglas D. Fang, Dajun Yang, Yifan Zhai. Predicting drug sensitivity to a novel BCL-2 and BCL-xL dual inhibitor in solid tumor PDX trials [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 2503.

Blockade of the checkpoint inhibitor programmed death 1 (PD-1) has gained big success in cancer therapy. However, the response rate of anti-PD1 agents remains low. Molecularly targeted agents offer selectivity and high tumor response rates, but patients develop resistance to these drugs inevitably. Combinations of targeted agents and immunotherapy provide new opportunities to improve cancer treatments. Recent studies found that p53 activation in the myeloid linage suppressed alternative (M2) macrophage polarization and attenuates tumor development and invasion, leading to the hypothesis that p53 activation may further augment antitumor immunity elicited by anti-PD-1 therapy. APG-115 is an orally active, selective, small molecule inhibitor of the MDM2-p53 protein-protein interaction. APG-115 acts as an antitumor agent by activating the p53 tumor suppressor in p53 wild-type tumors. However, its role in regulating immune responses remained unknown. In this study, we investigated the role of APG-115 in immune modulation both in vitro and in vivo. Enhanced antitumor activity was first demonstrated in p53 wild-type MH-22A, p53 mutant MC38, and p53 knockout (p53-/-) MH-22A syngeneic tumor models after the combination treatment of APG-115 and anti-PD-1 antibody. Despite differential changes in tumor-infiltrating leukocytes, including an increase in cytotoxic CD8+ T cells in the p53 wild-type tumors and an increase in proinflammatory M1 macrophages in the p53 mutant tumors, the combination treatment consistently reduced immunosuppressive M2 macrophages in the tumor microenvironment regardless of p53 status of tumor cells. In addition, in vitro, the treatment of bone marrow-derived macrophages with APG-115 resulted in activation of p53 and p21 gene expression, as well as a decrease in M2 macrophages population and reduction of c-MYC and M2-related gene expression. Moreover, enhanced M1 macrophage polarization in the spleen was also observed in naïve mice treated with APG-115. Furthermore, APG-115 increased production of multiple proinflammatory cytokines, including IFN-γ, TNF-α, IL-2 and IL-6, in stimulated T cells. Collectively, for the first time, our findings suggest that p53 activation by a pharmacological MDM2 inhibitor enables reversal of immunosuppressive tumor microenvironment and enhance antitumor immunity independently of p53 status of tumors. Specifically, in complementary to PD-1 blockade that predominantly activates cytotoxic CD8+ T cell populations, APG-115 primarily targets tumor-associated macrophages. Collectively, our data provide a rationale for applying the combination of APG-115 plus PD-1 blockade to a broader patient population with p53 mutant tumors. Accordingly, a clinical trial of APG-115 in combination with pembrolizumab in metastatic melanoma patients regardless of p53 status has been initiated in the USA. Citation Format: Douglas D. Fang, Qiuqiong Tang, Yanhui Kong, Qixin Wang, Jiaxing Gu, Xu Fang, Peng Zou, Tao Rong, Jingwen Wang, Dajun Yang, Yifan Zhai. Activation of p53 in the tumor microenvironment by MDM2 inhibitor APG-115 synergizes with PD-1 blockade independently of p53 status of tumor cells [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 3192.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are among the most prevalent B-lymphocyte neoplasms with Bruton’s tyrosine kinase (BTK) often found abnormally activated in these patients. BTK inhibitor ibrutinib has showed proven efficacy in both indications, but its clinical application is limited to gradually acquired resistance. Recent studies reported that ibrutinib-resistant cells exhibited higher BCL-2 expression and therefore increase their sensitivity to BCL-2 inhibitors. In this study, using a BCL-2 selective inhibitor APG-2575, we asked whether its combination with ibrutinib enhanced antitumor activity in FL and DLBCL in preclinical studies. Indeed, the combination treatment with APG-2575 and ibrutinib synergistically enhanced antitumor activity in DOHH2 cell-derived FL xenograft models. Similarly, enhanced activity was achieved in DLBCL xenograft models derived from OCI-LY1 cells. Interestingly, in OCI-LY1 xenografts, the combination treatment achieved a 100 % response rate, which include partially (PR) and complete tumor regression (CR), with ~80 % of CR and ~20 % of PR recorded. Durable response was continuously observed till the end of the study after dose suspension. Pharmacokinetic studies revealed that there were no significant changes in drug concentrations between single agents and the combination treatment, suggesting there was no drug-drug interaction between these two agents. On-target pharmacodynamic (PD) modulations of BTK and apoptosis pathways were observed in corresponding tumor tissues co-treated with ibrutinib and APG-2575. An increase in the levels of cleaved PARP-1 protein was observed under the combination treatment suggesting enhanced induction of apoptosis occurred, which may be the biological event contributing to the synergistic effect of the combination treatment. In summary, our study suggests that APG-2575 may be applied to the combination therapy with ibrutinib in the treatment of FL and DLBCL. Citation Format: Douglas D. Fang, Guoqin Zhai, Shoulai Gu, Ran Tao, Qiuqiong Tang, Ping Min, Qixin Wang, Dongmei Yang, Jiaxing Gu, Yinfeng Li, Dingxiong Chen, Jiajun Li, Guangfeng Wang, Dajun Yang, Yifan Zhai. BCL-2 selective inhibitor APG-2575 synergizes with BTK inhibitor in preclinical xenograft models of follicular lymphoma and diffuse large B-cell lymphoma [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 2058.

Focal adhesion kinase (FAK) plays an important role in cell migration, growth factor signaling, cell cycle progression and cellular survival. It had been shown that FAK and SRC family kinases were able to sustain downstream AKT and MAPK signaling under continuous EGFR inhibition driven by osimertinib (AZD9291), a mutant-selective third-generation EGFR inhibitor. Incomplete inhibition of AKT and MAPK was consistently observed even under increased concentrations of osimertinib and led to acquired resistance to osimertinib, implicating the need of combination therapy with FAK/SRC inhibitors to enhance antitumor activity of osimertinib and overcome its resistance. APG-2449 is a novel oral active small-molecular inhibitor that targets FAK, ALK and ROS1. In this study, we investigated the effect of combination treatment with APG-2449 and osimertinib using NSCLC NCI-H1975 cells carrying EGFRL858R/T790M mutation and an osimertinib-resistant patient-derived xenograft (PDX) model carrying EGFRT790M/19del/C797S mutation. In vitro, the combination treatment with APG-2449 and osimertinib synergistically inhibited the proliferation of NCI-H1975 cells. In vivo, APG-2449 significantly enhanced antitumor activity of osimertinib in NCI-H1975 xenograft models, leading to complete or partial tumor regression. More profound antitumor activity of this combination was also demonstrated in the osimeritinib-resistant PDX model in comparison with single agents. In term of mechanism, the combination arm significantly suppressed the on-target phosphorylation of EGFR, FAK and SRC, as well as AKT and ERK in NCI-H1975 xenografts. Collectively, these results suggest that addition of APG-2449 to osimeritinib may enhance antitumor activity and suppress the development of resistance in NSCLC. Citation Format: Guangfeng Wang, Douglas D. Fang, Ping Min, Ran Tao, Chunyang Tang, Shoulai Gu, Li Rui, Jiajun Li, Jingwen Wang, Miaoyi Wu, Yingfeng Li, Dingxiong Chen, Fei Zhang, Kejie Lian, Feifei Zhang, Lvcheng Wang, Rongcheng Xu, Dajun Yang, Yifan Zhai. Novel FAK/ALK/ROS1 inhibitor APG-2449 synergizes with osimertinib in preclinical xenograft models of EGFR-mutant NSCLC [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 2204.

Effective treatments are urgently needed for elderly acute myeloid leukemia (AML) and relapsed or refractory diffuse large B-cell lymphoma (DLBCL) patients as only 10-25% of the population respond to standard therapies and resistance often occurred. Concurrent overexpression of anti-apoptotic BCL-2 gene and proto-oncogene MYC are frequently found in AML and DLBCL and this molecular characteristic is associated with poor prognosis of patients. CDK9 controls non-ribosomal transcription of genes including MYC and MCL-1. Dysregulation in the CDK9 pathway had been reported in AML and DLBCL, making it an attractive target for the combination with BCL-2 inhibitor to achieve more effective therapy. To test the above hypothesis, in this study, we applied the combination of APG-2575 with a CDK9 inhibitor (alvocidib or dinaciclib) to preclinical tumor models. APG-2575 is a novel, orally bioavailable BH3-mimetic that selectively inhibits BCL-2, but not BCL-xL or MCL-1. After the treatment, synergistic effect of the combination treatment was demonstrated in mice bearing xenograft tumors of human AML or DLBCL. Specifically, in xenograft tumor models derived from human DLBCL cell line U2932, the treatment with APG-2575, alvocidib, or dinaciclib single agent for 22 days exhibited antitumor activity with T/C values of 20-50% (T/C value, average tumor volumes in treatment group vs. the vehicle control group). The combination treatment achieved substantial antitumor activity with T/C value of 5%. All animals in the combination groups showed either partial or complete tumor regression whereas none in the single arms. Similar synergistic antitumor activity of the combination treatment was consistently observed in the myelodysplastic syndrome SKM-1 and AML OCI-AML-3 xenograft models. Taken together, our data suggest that APG-2575 and CDK9 inhibition combination has a potential therapeutic application in treatment of patients with AML and DLBCL. Citation Format: Douglas D. Fang, Ran Tao, Qiuqiong Tang, Shoulai Gu, Guoqin Zhai, Jiajun Li, Qixin Wang, Xu Fang, Na Li, Dajun Yang, Yifan Zhai. Synergistic effect of BCL-2 inhibitor APG-2575 and CDK9 inhibitor in acute myeloid leukemia and DLBCL preclinical tumor models [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 3068.

Introduction: Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer-related mortality. Currently there are few treatment options available for advanced HCC patients. Multi-kinase inhibitors sorafenib and regorafenib are the only approved systemic HCC therapies and are only marginally effective in extending survival. HCC is largely driven by difficult-to-drug oncogenes, including β-catenin, and none of the current HCC therapeutics on the market or in development addresses these major genetic drivers of disease. β-catenin is an integral part of the WNT signaling pathway and plays a major role in regulation of cell survival, apoptosis and developmental processes. The goal of this study was to systematically interrogate contribution of β-catenin to HCC pathogenesis. Methods: The extent of genomic alterations in β-catenin itself or components of its regulatory network was interrogated in two large HCC data sets, The Cancer Genome Atlas and HCC cohort from Asan Medical Center. β-catenin in vitro credentialing (target knock-down, PD modulation and cell growth inhibition) was carried out in cultured HCC cell lines using multiple siRNAs against β-catenin. Stably expressed doxycycline-inducible shRNAs targeting β-catenin were used to establish in vivo dependency in HCC tumor xenografts. Results: Building on previous observations, we detected high frequency of somatic mutations in CTNNB1/β-catenin itself and in constituent members of its regulatory network in two independent cohorts of HCC patients. Furthermore, significant upregulation in β-catenin protein levels was detected in the overwhelming majority of HCC patient samples, patient-derived xenografts and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we discovered that dependency on β-catenin in human HCC cell lines generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1/β-catenin or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1/β-catenin and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in PD marker AXIN2, and decreased cancer cell proliferation. Conclusions: β-catenin is an important therapeutic target in HCC that could potentially be targeted using therapeutic siRNA suitably formulated for delivery to human liver tumors. Genomic alterations in CTNNB1/β-catenin, as well as increased abundance of non-phosphorylated (active) β-catenin, may serve as predictive biomarkers for patient selection. Citation Format: Zhihu (Jeff) Ding, Chaomei Shi, Lan Jiang, Tatiana Tolstykh, Hui Cao, Dinesh Bangari, Susan Ryan, Taiguang Jin, Mikhail Levit, Karl Mamaat, Qunyan Yu, Hui Qu, Joern Hopke, May Cindhuchao, Dietmar Hoffmann, Fangxian Sun, Mike Helms, Kerstin Jahn-Hofmann, Sabine Scheidler, Douglas Fang, Liang Schweizer, Jack Pollard, Christopher Winter, Dmitri Wiederschain. Sensitivity of liver cancer cell lines to B-catenin knock-down correlates with pathway activation [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 2489.

Background: Despite advances in the treatment of non-Hodgkin's lymphoma (NHL), refractory NHL remains a major clinical challenge. The B-cell lymphoma protein-2 (BCL-2) family is frequently overexpressed in NHL, which is associated with poor prognosis by promoting cell growth and resistance to antitumorigenic agents. In this study, by using different BCL-2 family protein inhibitors, we aim to further classify the survival dependency of NHL for anti-apoptotic BCL-2 family members and explore novel approaches to rationally develop combination therapies by completely blocking the activity of the Bcl-2 family proteins. Methods: Cell viability was determined by CCK-8 assay. Apoptotic activity was confirmed by Annexin V-propidium iodide (PI) apoptosis detection, Western blot analysis and mitochondrial membrane potentials detection analysis. In vivo, NHL xenograft models were established to analyze the antitumor effect of the BCL-2 family protein inhibitors. Results: We evaluated the inhibitory effect of of Bcl-2 targeted inhibitors and synergy in combination with other molecular targeted agents in NHL. While those NHL cell lines with only high levels of BCL-2 protein are sensitive to Bcl-2 selective inhibitor, the dual Bcl-2/Bcl-xL inhibitor APG-1252 and its active metabolite APG-1252-M1 potently induced mitochondria-dependent apoptosis in NHL cell lines that possess high expression of both BCL-2 and BCL-XL proteins. After the treatment of APG-1252-M1, apoptosis including caspase-3 activation, PARP cleavage, and mitochondrial membrane potentials change was observed rapidly within four hours. We further demonstrated that APG-1252-M1 showed synergistic effects with Bruton's tyrosine kinase (BTK) inhibitor ibrutinib in those sensitive cell lines by modulating STAT3 signaling pathways. However, cell lines harboring high expression of anti-apoptotic protein MCL-1 were resistant to APG-1252-M1. Furthermore, we observed that APG-1252-M1 exhibited strong synergistic effect with dual PI3K/mTOR inhibitors NVP-BEZ235 in those resistant cell lines with high levels of Mcl-2 protein. Conclusions: Taken together, our data provide an effective and precise therapeutic strategy for NHL through determining NHL addiction to anti-apoptotic BCL-2 family proteins, and then target-guided application of BCL-2 family inhibitors as a monotherapy or in combination with other molecular targeted agents to completely block the Bcl-2 family proteins, thus to restore the apoptotic cell death. Citation Format: Qiu-Yun Luo, Han-Jie Yi, Xiang-Lei Yan, Miaozhen Qiu, Bao-Xia Li, Lin Zhang, Wen-Tao Pan, Lu-Ping Yuan, Zhen-Yi Liu, Douglas D. Fang, Yifan Zhai, Jian Sun, Da-Jun Yang. Targeting the anti-apoptotic BCL-2 family protein provides an effective and precise therapeutic strategy for NHL [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 301.

Small cell lung cancer (SCLC) is one of the most deadly diseases with a dismal five year survival rate less than 7%. Even though most SCLC patients respond to the initial platinum-based cytotoxic or radiation therapies, they inevitably relapse and succumb to the disease. Anti-apoptotic proteins BCL-2 and BCL-xL, which are highly expressed in 40-60% of SCLCs and play a critical role in tumorigenesis and drug resistance, have been emerging as a promising target for therapeutic intervention. We have recently developed a novel dual BCL-2/BCL-xL inhibitor APG-1252 for cancer therapy. In this study, the effect of APG-1252 was evaluated in a panel of SCLC cell lines for discovery of indications and predictive biomarkers. The results show that the sensitivity of SCLC cell lines with sub-µM or nM IC50 values are correlated with the higher expression levels of BCL-2/BCL-xL, BIM and/or PUMA but lower levels of MCL-1. Conversely, the resistant cell lines either lack of BCL-2/BCL-xL protein, or exhibit higher level of MCL-1 protein. In xenograft tumor models, consistent with in vitro results, APG-1252 exhibits antitumor activities in the models derived from the sensitive cells but not in those from the resistant cells. Interestingly, while ABT-263 failed to inhibit H146 xenograft tumor growth, despite of its similar in vitro killing ability as APG-1252, APG-1252 showed potent antitumor activity in the xenograft model. To overcome the drug resistance conferred by MCL-1 in the resistant cells, we explore the combination therapy with other targeted agents. We found that our novel MDM2 inhibitor APG-115 was able to overcome the intrinsic resistance and sensitize those cells to APG-1252 in vitro, suggesting that reducing the apoptotic threshold by inhibiting other anti-death proteins like MCL-1 or increasing apoptotic function through p53 can enhance SCLC sensitivity to APG-1252. Collectively, APG-1252 represents a novel opportunity that can neutralize the protection from BCL-2/BCL-xL and trigger cell death and inhibit tumor growth in SCLC models. With the significance of these preclinical data, APG-1252 has been granted for phase 1 clinical trials in USA (NCT03080311) and China. Citation Format: GuangFeng Wang, Ping Min, MiaoYi Wu, Shuo Dang, ChuanYan Tang, Fei Zhang, Ming Guo, Shaomeng Wang, Jing Deng, Douglas D. Fang, DaJun Yang, YiFan Zhai. Targeting BCL-2 and BCL-xL with a novel dual inhibitor APG-1252 triggers cell death and inhibits tumor growth in small cell lung cancer models [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 307.

Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT. Besides surgical resection for primary localized tumors, imatinib remains the first-line treatment for advanced and metastatic GISTs. Imatinib targets a few kinases including KIT that often carries the primary driver mutations commonly located on exon 11 and exon 9. Unfortunately, a large proportion of the patients ultimately develop progressive disease owing to the secondary resistance mutations in KIT gene. We have developed an orally bioavailable multikinase inhibitor HQP1351, which is currently in clinical trials for the treatment of T315I mutant CML. Here, we demonstrated that HQP1351 inhibited both wild type and mutant (i.e., KITL576P, KITV559D, and KITV559D/T670I) KIT in biochemical assays. Using a panel of imatinib-resistant and sensitive GIST cancer cell lines derived from patient samples, we showed that HQP1351 exhibited more potent anti-proliferative activities than ponatinib (range of IC50 values: 0.027-0.133 µM vs. 0.021-0.730 µM), the latter of which is currently in clinical development in GIST to overcome the resistance to imatinib (NCT03171389). In addition, the treatment of GIST cancer cells with HQP1351 in vitro led to more profound inhibition of pharmacodynamic markers, including p-c-KIT, p-AKT, p-ERK1/2, and p-AKT, in comparison with ponatinib. Correspondingly, in multiple xenograft tumor models derived from these GIST cancer cell lines carrying the secondary mutations of KIT, HQP1351 exhibited superior antitumor activities to ponatinib. Collectively, considering that HQP1351 has already been in clinical trials, the above results suggest that therapeutic application of HQP1351 in imatinib-resistant GIST patients deserves further investigation in human. Citation Format: Guangfeng Wang, Haibo Qiu, Ping Min, Miaoyi Wu, Shuo Dang, Chunyang Yang, Fei Zhang, Wei Zhuang, Zhiwei Zhou, Douglas D. Fang, Dajun Yang, Yifan Zhai. HQP1351, a novel multikinase inhibitor in clinical development, overcomes drug resistance for the treatment of gastrointestinal stromal tumors in preclinical models [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 1979.

Recent studies suggest that cancer can arise from a cancer stem cell (CSC), a tumor-initiating cell that has properties similar to those of stem cells. CSCs have been identified in several malignancies, including those of blood, brain, and breast. Here, we test whether stem cell–like populations exist in human melanomas. In ∼20% of the metastatic melanomas cultured in growth medium suitable for human embryonic stem cells, we found a subpopulation of cells propagating as nonadherent spheres, whereas in standard medium, adherent monolayer cultures were established. Individual cells from melanoma spheres (melanoma spheroid cells) could differentiate under appropriate conditions into multiple cell lineages, such as melanocytic, adipocytic, osteocytic, and chondrocytic lineages, which recapitulates the plasticity of neural crest stem cells. Multipotent melanoma spheroid cells persisted after serial cloning in vitro and transplantation in vivo, indicating their ability to self-renew. Furthermore, they were more tumorigenic than adherent cells when grafted to mice. We identified similar multipotent spheroid cells in melanoma cell lines and found that the stem cell population was enriched in a CD20+ fraction of melanoma cells. Based on these findings, we propose that melanomas can contain a subpopulation of stem cells that contribute to heterogeneity and tumorigenesis. Targeting this population may lead to effective treatments for melanomas.

Hepatocellular carcinoma (HCC) is the fifth most common type of cancer worldwide. However, current therapeutic approaches for this epidemic disease are limited and its 5-year survival rate has not been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited point mutations in JAK1 (Janus Kinase) gene, which were confirmed by Sanger sequencing. To explore the transforming capability, these mutations were then introduced into 293T and Ba/F3 cells by transient transfection and retroviral infection, respectively. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore, a modest sensitivity to the treatment of a JAK1/2 inhibitor, Ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly inhibited. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo. JAK-STAT pathway might represent a new therapeutic opportunity for HCC. Monotherapy using a more potent inhibitor and combinatory therapy may be further explored in JAK1S703I mutant PDX model. Citation Format: Shuqun Yang, Chonglin Luo, Qingyang Gu, Qiang Xu, Hongye Sun, Ziliang Qian, Yexiong Tan, Hao Wu, Yuxin Qin, Yuhong Shen, Xiaowei Xu, Shu-Hui Chen, Chi-Chung Chan, Hongyang Wang, Mao Mao, Douglas D. Fang. Activating JAK1-S703I mutation may predict the sensitivity of JAK-STAT inhibition in hepatocellular carcinoma patient-derived xenograft tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 688. doi:10.1158/1538-7445.AM2015-688

Targeted drugs have been widely used in the treatment of lung cancer, especially for non small cell lung cancer (NSCLC) patients. NCCN guideline lists multiple drugs which are particularly effective in the subgroup of patients with specific gene mutation variants, including the fusion transcripts of ALK, ROS1 and RET, and the mutations of EGFR, KRAS, BRAF and HER2. Multiple techniques have been developed to detect fusion transcripts in tumor samples, such as FISH, IHC and PCR assays. FISH is the most accurate method for fusion detection currently. The split probe FISH assay, which does not require the pre-knowledge of what is the fusion partner with ALK for example, can detect unknown ALK fusion products as well. However, it fails to identify which gene is fused with ALK (or other oncogene) in the chimera products, therefore is not informative for the mechanism or drug resistance study. In addition, due to the limitation on the fluoresce dye, it is quite difficult to multiplex fusion FISH probes for different genes into one assay. We describe here a next generation sequencing assay to detect a panel of fusion transcripts and mutations in one single assay. Our single assay can detect the fusion products of ALK, ROS1 and RET with any known or unknown fusion partners, together with the hotspot mutations in EGFR, KRAS, BRAF and HER2. Our assay can identify not only the exact sequence of the unknown fusion partners, but also any mutations within the full coding regions of ALK, ROS1 and RET. This information is critical for the resistance mechanism study and the next generation inhibitor drug development, since about half of the ALK inhibitor resistance was caused by the mutations in ALK gene itself. We have identified a novel ALK fusion gene with a never-reported fusion partner in a lung cancer patient. The xenograft model derived from this patient responds to crizotinib well in the in vivo study (published on Journal of Thoracic Oncology). We have demonstrated that our assay can reliably detect both the novel fusion and the well known EML4-ALK fusion from only 1-2 5um FFPE slides of the lung tumor tissues, together with other possible fusions and mutations as well. Note: This abstract was not presented at the meeting. Citation Format: Qiang Xu, Guan Wang, Bo Li, Maoxiang Qian, Douglas Fang, Lele Sun, Yundi Chen, Hongye Sun. A next generation sequencing assay to detect the fusion products of ALK, ROS1 and RET with any fusion partner genes and hotspot mutations in FFPE samples. [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 4668. doi:10.1158/1538-7445.AM2014-4668

Hepatocellular carcinoma (HCC) is intrinsically resistant to conventional chemotherapies. Developing effective therapeutics becomes critical to tackle the unmet medical need. The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling axis plays an important role in liver development and homeostasis in adults. Deregulation of the FGF/FGFR signaling pathway through genetic modification or overexpression of the receptors (or their ligands) has been increasingly recognized in HCC. Accordingly, therapeutic intervention of the pathway has been extensively explored. In this study, analysis of a panel of patient-derived xenograft (PDX) HCC models by Affymetrix human gene expression arrays revealed overexpression of FGFR1 in 13 out of 43 (30%) models. Antitumor activities of FGFR inhibitors, including lenvatinib, dovitinib, ENMD-2076, and LY2874455, were then tested in five models. Lenvatinib exhibited the most potent activity in all models, whereas LY2874455 failed to show the effect in one of models. IC50s for FGFR1 inhibition of ENMD-2076, lenvatinib, dovitinib and LY2874455 were 145 nM, 134.5 nM, 53.5 nM and 0.6 nM, respectively. In SNU-16 and NCI-H520 cell lines, IC50s of dovitinib, lenvatinib, ENMD-2076, and LY2874455 were 76/1355 nM, 94/4770 nM, 306/2280 nM, and 0.3/664 nM, respectively. Furthermore, analyses of PDX tissues by Affymetrix SNP Array 6.0 arrays revealed no amplification of FGFR1/2 genes in four models except for amplifications of FGFR3 gene in two models. Whole exome sequencing did not detect nonsynonymous genetic alterations, including SNPs and indels, in FGFR1/2 genes in these xenografts except for a few genetic variations in FGFR3/4 genes. Overall, due to the multi-targeting mechanisms of these inhibitors, in vitro inhibitory effects of the compounds on FGFR1 enzyme and cell proliferation may not correlate with the in vivo anti-tumor activities. Overexpression of FGFR1 gene may serve as a predictive biomarker for therapeutic intervention of the FGF/FGFR pathway in HCC by using lenvatinib, dovitinib and ENMD-2076. Further studies are required to define additional biomarkers for LY2874455. Thus, our findings provide the rationale for the clinical development of FGFR inhibitors in a subset of HCC patients expressing a high level of FGFR1 gene. Citation Format: Douglas D. Fang, Bin Zhang, Weiguo Xu, Kang Yan, Qingyang Gu, Qiang Xu, Yexiong Tan, Hongye Sun, Qin Luo, Weifeng Mao, Chiho Li, Jiangpeng Liao, Guibai Liang, Shu-Hui Chen, Chi-Chung Chan. Antitumor activities of FGFR inhibitors in FGFR1-overexpressing hepatocellular carcinoma patient-derived xenograft tumor models. [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 2839. doi:10.1158/1538-7445.AM2014-2839

Ovarian cancer has the highest mortality rate of all gynecological cancers primarily due to the high incidence of metastatic spread prior to diagnosis. Understanding the biology of this malignancy and developing models that reflect the patient disease phenotype will create better pre-clinical opportunities for testing targeted therapy. We used the Ovcar 3 cell line to establish a metastasis model through serial in vivo passage. The dual PI3K/mTOR inhibitors PF-04691502 and PF-05212384 were then investigated in this model for their therapeutic effect against metastatic disease progression. Notably, the stromal environment and tumor initiating subpopulation play critical roles on the Ovcar 3MET disease invasiveness and progression. Based on the molecular profile, these invasive Ovcar 3MET cells exhibited a more EMT phenotype when compared to parental cells. Impairing PI3K/mTOR pathway with the dual PI3K/mTOR inhibitors resulted in significant anti-metastatic activity and increased survival in the Ovcar 3MET model. Serum CA125 level, which is a validated clinical marker, correlated with anatomic measurement of tumor burdens via BLI and mouse survival time in these studies, providing means for longitudinal monitoring of disease progression and response to therapy. In addition, using ex vivo tumorsphere analysis under serum-free condition, both PF-04691502 and PF-05212384 demonstrated robust activity against the self-renewal ability of Ovcar 3 tumor cells. In summary, this translational tumor model may serve as a valuable tool for testing targeted therapies such as PI3K/mTOR inhibitors for their ability to impact progression of metastatic ovarian disease. Citation Format: Zhengming Yan, Douglas Fang, Brett Simmons, Jing Yuan, Julie Kan, Cathy C. Zhang. Dual targeting of the PI3K/mTOR pathways by PF-04691502 or PF-05212384 exhibits therapeutic effects against the metastatic disease progression in the Ovcar 3 disseminated model. [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 322. doi:10.1158/1538-7445.AM2013-322

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer-related deaths. Half of the total HCC-related deaths and new cases occurred in China. Different from many cancers, HCC is intrinsically resistant to conventional chemotherapies, and many attempts to develop new systemic agents for HCC have failed in clinic. Lack of clinically relevant and genetically well-defined preclinical models critically accounts for the failures. In this study, we established an extensive panel of Chinese HCC patient-derived xenograft (PDX) models by directly transplanting surgically resected patient tumor tissues in BALB/c nude or SCID mice. Implantation of 254 patient samples resulted in 70 transplantable PDX models (27.6%) covering stage I, II, III, and IV tumors. Xenograft tissues could be serially passaged, cryopreserved, and revived with a success rate of over 94%. Early passages between p2-p4 of PDX tissues were used in the histological and molecular characterization. The histology of PDX tissues recapitulated the features of the original samples and maintained the heterogeneity of human tumors. Unsupervised hierarchical cluster analysis of gene expression profiles in 7 paired samples assessed by GeneChip® PrimeView™ Human Gene Expression Array revealed that PDX tumor tissues retained the global gene-expression patterns of patient samples. Using next-generation sequencing we performed exome sequencing of 43 of PDX models. Contaminated murine DNA reads were removed by a proprietary algorithm to eliminate homology bias. A wide variety of SNPs were identified and some may represent driver mutations, such as ones on JAK1 and JAK2 genes. Copy number variation analysis by Genome-Wide Human SNP Array 6.0 in 24 PDX models elucidated amplifications of a number of genes, including MET, AKT2, AKT3, mTOR, ALDH1A1, ALDH1A2, JUN and NOV. In vivo efficacy studies exhibited differential responses to standard-of-care compounds in the models. Collectively, with the current intense interest in development of targeted therapies, the use of patient liver cancer tissue-derived models with well genomic characterization provides an excellent, and perhaps more relevant, platform for preclinical studies of experimental therapeutics, and for biomarker discovery and translational research. Citation Format: Douglas D. Fang, Qiang Xu, Yexiong Tan, Lei Chen, Hongye Sun, Qingyang Gu, Bin Zhang, Qin Luo, Shu-hui Chen, Hongyang Wang, Chi-Chung Chan. Genetic characterization of an extensive panel of patient-derived xenograft tumor models of hepatocellular carcinoma for preclinical development. [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 2739. doi:10.1158/1538-7445.AM2013-2739

The genomes of all cancer cells carry somatic mutations, including both passenger and driver mutations. Driver mutations, which are implicated in oncogenesis, are most likely preserved in xenografted tumor cells in immunedeficient mice. Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas in the gastrointenstinal tract. It is prevalent in the developing world and associated with substantial morbidity and mortality. Molecular mechanisms contributing to initiation and progression of ESCC are still poorly understood. Systematic identification of genetic variations and putative driver mutations may lead to understanding of pathogenesis of the disease. In this study, using next-generation sequencing we performed exome sequencing of eight paired Chinese esophageal squamous cell carcinoma and xenograft. Comparing with control genomes obtained from case-matched peripheral blood cells, we identified a wide variety of somatic variations in both primary and xenograft tumors, including TP53, RGMA, MUC4, TCEAL6, PRSS3, etc. Moreover, we found that mouse sequences accounted for a significant fraction of sequence reads from xenograft samples. Excluding these mouse sequences significantly improved the accuracy of mutation detection in xenograft samples. Overall, our findings shed a light on important somatic mutations involved in ESCC. In addition, comparison between primary and xenograft tumors may lead to identification of driver mutations that confer growth advantage. These mutations represent therapeutic opportunities for development of targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2351. doi:1538-7445.AM2012-2351

Notch signaling is known to be a survival pathway for tumor-initiating cells. In this report, we demonstrate that the γ-secretase inhibitor PF-03084014 significantly enhances the antitumor activity of docetaxel in multiple xenograft models of triple-negative breast cancer. Mechanistic evaluation revealed that PF-03084014 perturbs the Notch signaling pathway and suppresses the function of tumor initiating cells (TIC). In MDA-MB-231Luc model, treatment of docetaxel led to a significant increase of CD133+/CD44+ and ALDH+ subpopulations by FACS analysis. In combination with PF-03084014, these two unique cell subpopulations were significantly diminished. Correspondingly, the functional analyses by tumor re-implant and mammosphere-forming efficiency assays revealed that docetaxel-therapy promoted the tumor initiating capability of the remaining cells, in which an increased stem cell property and Notch pathway activation were observed through gene signature changes. In contrast, PF-03084014 co-treatment with docetaxel substantially hampered the self-renewal ability of these cells. Notch target gene analysis demonstrated the biological relevance of PF-03084014-induced activity. To characterize the function of CD133+/CD44+ subpopulation, MDA-MB-231Luc tumors were de-bulked by the treatment with docetaxel. Subsequently, the CD133+/CD44+ and CD133-/CD44- subpopulatons were isolated and re-implanted in SCID-bg mice using a limiting dilution approach. The results showed that CD133+/CD44+ cells gave rise to tumors with a 100 % take rate (10/10), whereas CD133-/CD44- cells were not tumorgenic (0/10). In addition, CD133+/CD44+ cells exhibited much higher tumorigenicity compared with the respective adherent parental cell line. PF-03084014 treatment caused a significant delay of CD133+/CD44+ tumor growth. The ability of PF-03084014 to suppress TICs was also observed in other breast cancer xenografts, including patient derived models. This data suggests that anti-TIC is one of the contributing mechanisms for the synergistic activities of PF-03084014 in combination with docetaxel. Our work provides potential therapeutic opportunities for PF-03084014 to improve conventional cytotoxic therapy by inhibiting Notch signaling in tumor-initiating cells and other bulk tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3492. doi:1538-7445.AM2012-3492

The double hits theory suggests a major mechanism of tumorigenesis. However, compared to the somatic mutations, the loss of heterozygosity (LOH) was not frequently identified in many sequencing projects. Unlike the somatic mutations that were detected by the evidence of novel alleles in tumor tissue, the alleles involving in the LOH are present in normal tissues as well. Given the high level of tissue heterogeneity of primary tumors, including both tumorous cells and normal stroma cells, at whole tissue or million cells level LOH can only be detected as a quantitative change between tumor and normal samples, which are not easily distinguished from potential sequencing errors or other artifacts especially with low depth sequencing. By applying whole exome capture deep sequencing on case-matched primary tumor, blood and xenograft samples, we found that xenografts represent a much purer human tumor cell population than primary tumor tissues. These tumor cells growing in mice not only contain the majority of somatic mutations in the primary tumors (>95%), but also reveal a >20 folds enrichment of somatic LOH events, which were not categorized as LOH in primary tumors, versus somatic mutations. All these findings were detected after removing mouse sequence contaminations, which if included will induce about half of the total variations observed in the xenograft samples. We found that these LOH events affect both tumor suppressor genes and oncogenes. Given that driver mutations or other variations that are implicated in oncogenesis are most likely preserved in xenografted tumor cells in immunedeficient mice, we hypothesize that the bigger range of somatic LOH events that we observed in xenograft samples play a potential ever important role in tumorigenesis than what we thought previously. Our finding also sheds a new light on a novel approach for the discovery of potential drug targets by sequencing xenograft samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-397. doi:1538-7445.AM2012-LB-397

Aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in several human cancers. Mutations in the Patched (PTCH1) gene are responsible for basal cell nevus syndrome, and are commonly found in sporadic basal cell carcinoma and in medulloblastoma. In this study we evaluated PF-04449913, an inhibitor of the Hh signaling pathway, in a Ptch1+/-p53 mouse model of medulloblastoma and in human patient derived xenograft models. Treatment of Ptch1+/-p53+/- or Ptch1+/-p53-/- medulloblastoma allografts with PF-04449913 produced potent dose-dependent inhibition of Hh pathway activity resulting in stable tumor regression. Using Gli1 transcript levels as a surrogate for Hh pathway activity, the pharmacodynamic effects of PF-04449913 were evaluated in medulloblastoma allografts following single dose and multi dose administrations of compound. PF-04449913 treated medulloblastoma allografts had reduced levels of Gli1 gene expression and down regulation of genes linked to the Hh signaling pathway. PF-04449913 was also effective when combined with a chemotherapeutic agent in a colon patient derived xenograft model and a pancreatic patient derived xenograft model, resulting in 63% and 73% tumor growth inhibition respectively. Collectively, our study demonstrates the therapeutic efficacy of a small molecule inhibitor of Hh pathway in preclinical models of multiple cancer types in either single or combination treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4504. doi:10.1158/1538-7445.AM2011-4504

RON, the receptor for macrophage-stimulating protein, belongs to the MET receptor tyrosine kinase family and shares many common features with MET. Inappropriate activation of RON signaling plays an important roll in colon carcinoma cell proliferation, survival, migration and invasive growth. Ron and Met are overexpressed in patient colon tumors, where co-overexpression of both Ron and Met correlates with the worst patient survival compared to the overexpression of Ron or Met alone. BRaf-activating mutations occur in about 10% of colorectal carcinomas. Mutant BRaf has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase (MAPK) pathway, and BRaf mutant cancers are generally resistant to standard therapies. Here we report that knockdown of the Ron gene by Ron shRNA in BRafV600E mutant HT29 colon carcinoma cells induced HT29 tumor apoptosis and resulted in significant tumor growth inhibition (TGI) by 59%, which is comparable to that observed with the selective EGFR inhibitor-Erlotinib treatment (57% TGI), but more potent than that of the selective Met inhibitor-PF-04217903 treatment (38% TGI). When Ron shRNA knockdown and treatment with the selective Met inhibitor were combined, enhanced tumor growth inhibition (76%) was observed. Comparable experiments using the BRaf mutant Colo205 model yielded similar results. Western Blot analysis showed that combination of Ron shRNA and Met inhibition in HT29 cells decreased phosho-ERK, elevated cleaved caspase-3 and, surprisingly, increased total Ras protein levels. No significant changes in total and phospho-AKT levels were observed. In vitro, the stable HT29-shRon cells initially grew more slowly compared to HT29-vector cells, but the growth rate gradually increased, approaching that observed in normal cell culture conditions. Interestingly, when co-culturing stable HT29-shRon or HT29-vector cells with bone marrow derived mesenchymal stem cells, the selective Met inhibitor-PF-04217903 induced significant cell death only in the HT29-shRon cells under hypoxic conditions (1% oxygen). No cell death was observed with either HT29-shRon cells under normoxia or HT29-vector cells under hypoxia or normoxia conditions, indicating the collaborative roll of Ron and Met in colon cancer cell survival during hypoxia. In summary, inhibiting both Ron and Met pathways in BRaf mutant colon cancer cells down- regulated ERK activity, induced apoptosis and led to enhanced antitumor efficacy in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5409.

Emerging evidences suggest that cancer stem cells (CSC) may be critically responsible for tumor initiation, progression, metastasis, and drug resistance. It becomes important to ask whether anti-cancer agents are able to target the tumor-initiating subpopulation in relevant CSC models. In this study, we first established xenograft tumors in NOD/SCID mice from a colorectal cancer patient specimen and demonstrated that CD133/EpCAM-expressing CSC population was highly tumorigenic. We then sought to propagate the CSC population under a serum-free condition. In culture, the tumor cells formed non-adherent, 3-dimensional spheroids, a fraction of which retained expression of the CSC markers. When exposed to a serum-containing medium, tumor spheroid cells differentiated into epithelial-like adherent cells with an increase in cell proliferation rate. In comparison with the differentiated progeny, tumor spheroid cells exhibited resistance to the standard-of-care agent oxaliplatin and, in limiting dilution assays in mice, displayed substantially higher tumorigenic potential. In contrast to the tumors originated from the differentiated cells, tumor spheroid cell-derived tumors recapitulated not only the CSC frequency marked by CD133/EpCAM expression, but also the histological characters of the original tumor. Similarly, only were the fragments of spheroid cell-derived xenograft tumors capable of regenerating highly proliferative tumors in secondary transplantation. Thus, the tumor spheroid culture is indeed enriched of drug resistant, self-renewing, and tumor-initiating CSC populations. Mutation profiling of frequently mutated oncogenes using Sequenom OncoCarta™ panel identified a mutation in the kinase domain of PIK3CA (H1047R) in the cultured CSCs. This mutation has been reported present in a large number of colon cancer patients and likely functions as an oncogene (Samuels et al., Science 304:554; 2004). We further demonstrated that a dual mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitor (PF-04691502) exhibited a more potent effect on inhibition of in vitro proliferation of the mutated CSCs compared to the chemotoxic agent oxaliplatin. Collectively, our findings suggest that CSC models provide a novel avenue to drug sensitivity and efficacy studies. The well-characterized CSC model systems may assist in the development of more effective therapy against the subpopulation of tumors driven by the CSCs bearing specific mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4483.

We have demonstrated previously that L-histidinol, a structural analogue of the essential amino acid L-histidine, protects a variety of phenotypically normal cell lines from certain proliferation-dependent anticancer drugs without decreasing the toxicity of these agents for corresponding tumorigenic derivatives of the normal cells. Histidinol modulates the toxicity of selected anticancer drugs in tissue culture systems by its ability to arrest, specifically and reversibly, cells of normal phenotype in a G0-like, noncycling state while allowing continued cell cycle transit in most of their tumorigenic counterparts. Thus, in the presence of comparable levels of histidinol, the toxicities of the proliferation-dependent anticancer drugs 1-beta-D-arabinofuranosylcytosine and 5-fluorouracil are eliminated for a variety of normal cell lines but significantly increased for a number of tumorigenic lines. We report here that histidinol confers substantial protection upon the bone marrow cells of DBA/2J mice from the drugs 1-beta-D-arabinofuranosylcytosine and 5-fluorouracil. The protective responses were evaluated by quantitative cell survival assays and by animal survival studies. We report also that the histidinol-mediated protection to bone marrow cells persists in L1210 leukemia-bearing DBA/2J mice treated with combinations of histidinol and 1-beta-D-arabinofuranosylcytosine or 5-fluorouracil without diminishing the toxicities of these agents for in situ leukemia cells.