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mTOR Ser/Thr protein kinase operates in two distinct multi-protein complexes, TORC1 and TORC2, which together regulate growth, metabolism, angiogenesis and survival by integrating nutrient and hormonal environmental signals. The activity of mTOR is frequently up-regulated in human cancer by constitutive mitogen stimuli or oncogenic mutations upstream of TORC1 and TORC2. Rapamycin provides mechanistic rationale and clinical proof of concept for the therapeutic value of targeting mTOR in human cancer, but it also provided insights into how ATP-competitive TORC1/2 inhibitors have the potential to demonstrate superior efficacy. Through rational drug design we have discovered INK128, a potent, selective TORC1/2 inhibitor with excellent drug-like properties. INK128 inhibits mTOR kinase (sub-nanomolar) in an ATP-dependent fashion and demonstrates a high degree of selectivity against closely related kinases as well as against a panel of more than 400 kinases. INK128 inhibits both the phosphorylation of S6 and 4EBP1, the downstream substrates of TORC1, and selectively inhibits AKT phosphorylation at Ser473, the downstream substrate of TORC2, in vitro and in vivo. Interestingly, potent inhibition was also observed in cell lines resistant to rapamycin and pan-PI3K inhibitors. Daily, oral administration of INK128 (alone or in combination) inhibited angiogenesis and tumor growth in multiple xenograft models with predicted dose:exposure PK/PD relationship. We attribute the superior activity of INK128 to the fact that it is a more effective inhibitor of TORC1 relative to rapamycin. We further demonstrated that TORC1/2 inhibitors cause death of murine and human leukemia cells in models of pre-B acute lymphoblastic leukemia. In vivo, oral daily treatment with TORC1/2 inhibitors delayed leukemia onset and augmented the effects of ABL kinase inhibitors. Unexpectedly, these novel TORC1/2 inhibitors had much weaker effects than rapamycin on proliferation and function of normal lymphocytes. These findings establish that transformed lymphocytes are selectively sensitive to active-site TORC1/2 inhibitors and further support the development of such compounds for leukemia therapy in addition to solid tumors. In summary, INK128 is a potent, selective, and orally active TORC1/2 dual inhibitor positioned to enter clinical development. TORC1/2 inhibitors are mechanistically distinct from rapamycin and offer a compelling approach to the treatment of cancer by targeting translational control, cell metabolism, growth and angiogenesis. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B148.

Background: The PI3K pathway is one of the most frequently dysregulated pathways in human cancer. Activating mutations in the PIK3CA gene, encoding the p110 catalytic subunit of PI3K, have been identified as a major mechanism of inducing oncogenic PI3K signaling. The high frequency of PIK3CA mutations suggests that PI3K inhibitors may have therapeutic utility in genetically defined tumor populations. The discovery of isoform-selective PI3K inhibitors has proven to be difficult due to the highly homologous nature of all PI3K isoforms and structurally related kinases. PI3Kα-selective inhibitors may permit more potent inhibition of PI3Kα while minimizing side effects and permitting more alternatives for combination therapy relative to non-selective class I PI3K (pan-PI3K) pathway inhibitors, many of which have entered clinical development. Results: Through structure-guided drug design we have discovered INK1117, a novel, potent and selective PI3K inhibitor with good oral bioavailability. INK1117 potently inhibits PI3K and demonstrates a greater than 100-fold selectivity relative to other class I PI3K family members and mTOR as well as a high degree of selectivity against a large panel of protein kinases. INK1117 blocks proliferation of tumor cell lines bearing PIK3CA mutations, and inhibits cellular phosphorylation and activity of AKT. However, INK1117 shows much less activity in PTEN-deficient tumor cells, which typically display constitutive PI3K pathway activation independent of PI3Kα. Daily, oral administration of INK1117 potently inhibits tumor growth in xenograft models bearing PIK3CA oncogenic mutations and, comparable to in vitro studies, INK1117 was not efficacious in tumor models with PTEN and/or KRAS mutations. In contrast to pan-PI3K inhibitors, INK1117 does not induce significant glucose elevation in glucose unchallenged or challenged rodents while levels of circulating Insulin is mildly elevated. The absence of hyperglycemia but presence of moderate hyperinsulinemia in mice treated with INK1117 is in line with results obtained using genetically engineered PI3K deficient mice. Additionally, INK1117 does not significantly impair B and T cell function in vitro and in vivo. INK1117 in combination with targeted agents such as lapatinib or trastuzumab, displayed enhanced activity both in vitro and in vivo. Our preclinical data show that PI3K isoform-selective kinase inhibitors as a single agent can provide comparable or superior efficacy than panPI3K inhibitors in tumors with PIK3CA mutation with better tolerability. The combination of PI3K inhibitors with other oncogene-targeted drugs revealed further improved anti-tumor efficacy. Conclusion: Selective targeting of PI3K combined with companion diagnostic presents a novel therapeutic strategy for patients with PIK3CA mutated cancer types. INK1117 is a potent and orally efficacious PI3Kα-selective kinase inhibitor currently in phase 1 clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A171.

CRPC is associated with primary and acquired chemotherapy resistance. Loss of androgen receptor (AR) signaling or development of AR splicing variants (e.g., ARV7) in CRPC imparts resistance to standard of care (SOC) agents that target AR signaling (e.g., enzalutamide and abiraterone). Effective therapies are an unmet need for CRPC patients with treatment-resistant tumors. Aberrant Wnt pathway activation contributes to resistance to AR-targeted agents, and cytotoxic chemotherapies such as docetaxel have been shown to activate Wnt signaling in PC cells. SM08502 has demonstrated strong antitumor activity in several preclinical cancer models and has been shown to inhibit the Wnt signaling pathway via disruption of alternative splicing. Here, we examined the antitumor activity of SM08502 in preclinical models of CRPC. The effect of SM08502 on cell proliferation was tested in 5 PC cell lines. Proliferation was strongly impaired by SM08502 across all cell lines (average EC50=0.319 μM [0.191–0.462]) irrespective of their mutation profile or hormone sensitivity. Compared to DMSO, SM08502 inhibited serine/arginine-rich splicing factor 6 (SRSF6) phosphorylation and potently suppressed Wnt-related gene (LRP5, TCF7, TCF7L1) and protein expression. In vivo antitumor effects and tolerability of QD oral SM08502 were assessed in multiple xenograft models, including mice bearing 22RV1 (ARV7+) or PC3 (AR-/-) CRPC flank xenografts (n=6/group). In 22RV1 xenografts, tumor growth inhibition (TGI) was demonstrated in mice treated with 12 and 25 mg/kg SM08502 (35%, P<0.05; 73%, P<0.001, respectively) vs. vehicle at D24 of treatment. In PC3 xenografts, significant TGI was seen in mice treated with 25 mg/kg SM08502 (75%, P=0.03) vs. vehicle. In 22RV1 xenografts, no TGI was observed with 75 mg/kg abiraterone or 30 mg/kg enzalutamide treatment (-4% and 12%, respectively) vs. vehicle, which confirmed the effect of ARV7 on resistance. Similarly, no significant TGI was seen in PC3 xenografts treated with enzalutamide, abiraterone, or docetaxel (-52%, P=0.2; -60%, P=0.33; 26%, P=0.38, respectively). No combination effect on TGI was observed with 25 mg/kg SM08502 + docetaxel (90%, P<0.05), but more tumor regressions occurred with combined treatment (4/6) than SM08502 alone (2/6). SM08502 was well tolerated in all tested xenograft models (<15% bodyweight loss from baseline). In summary, SM08502 potently inhibited cell proliferation, SRSF6 phosphorylation, and Wnt-related gene expression in multiple PC cell lines. In vivo, SM08502 demonstrated strong antitumor effects in CRPC xenografts. These data suggest that SM08502 has the potential to provide clinical benefit to patients with treatment-resistant CRPC. A Phase 1 study of SM08502 in subjects with advanced solid tumors is ongoing (NCT03355066). Citation Format: Carine Bossard, Nathalia Cruz, Kevin Chiu, Brian Eastman, Chi Ching Mak, Sunil KC, Gail Bucci, Josh Stewart, Timothy J. Phalen, Steven Cha. SM08502, a novel, small-molecule CDC-like kinase (CLK) inhibitor, demonstrates strong antitumor effects and Wnt pathway inhibition in castration-resistant prostate cancer (CRPC) 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 5691.

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD) mutation accounts for ~25% of all AMLs, carries a poor prognosis, and is prone to relapse despite targeted therapy. FLT3 mutations are associated with aberrant activation of the Wnt signaling pathway, which itself is implicated in AML initiation/progression and is required for the self-renewal and survival of leukemic stem cells. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway. These studies examined the antitumor activity of SM09419 as a single agent and in combination with targeted and standard therapies in preclinical models of FLT3-ITD AML. In MV-4-11 and MOLM-13 AML cells carrying the FLT3-ITD mutation, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt pathway-related genes (CCND1, MYC, TCF7, DVL2). The effect on cell proliferation was tested in 8 AML cell lines with varying mutation profiles as well as 26 different leukapheresis-derived primary human AML cells. Proliferation was strongly impaired by SM09419 across all tested cell lines (average EC50=0.2 + 0.048 µM]); MV-4-11 and MOLM-13 cells had EC50 of 0.049 and 0.144 µM, respectively. SM09419 also potently inhibited proliferation in all primary AML cells (average EC50=0.048 + 0.0097 µM) regardless of FLT3 mutation status, cytogenetics, or AML diagnosis (de novo or relapsed/refractory). SM09419 also induced apoptosis in MV-4-11 and MOLM-13 cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression relative to vehicle. In vivo antitumor effects and tolerability of oral SM09419 (QD) alone or combined with either midostaurin (FLT3 inhibitor) or venetoclax (BCL2 inhibitor) and/or azacitidine were assessed in FLT3-ITD xenograft models (n=5-6/group). In MOLM-13 xenografts, SM09419 (12.5 and 25 mg/kg) induced strong tumor growth inhibition (TGI) vs. vehicle at Day 14 (TGI 52% [p<0.05] and 74% [p<0.001], respectively). Midostaurin (50 mg/kg) induced significant TGI vs. vehicle (50%, p<0.05), which was increased when administered in combination with 12.5 mg/kg SM09419 (81%, p<0.001). In MV-4-11 xenografts, single-agent SM09419 (6.25, 12.5, and 25 mg/kg) induced significant TGI vs. vehicle (56% [p<0.05], 94%, and 95% [p<0.001], respectively) at Day 26 with tumor regression in all mice dosed at 12.5 mg/kg and 25 mg/kg. In a subsequent experiment, midostaurin (50 mg/kg) alone and combined with 6.25 mg/kg SM09419 for 23 days induced tumor regression in MV-4-11 xenografts (100% TGI vs. vehicle, p<0.0001). After treatment discontinuation, tumor regression was maintained in all mice (6/6) treated with the combination for 26 days, whereas tumor regrowth was immediately observed in midostaurin-treated mice. In another MV-4-11 xenograft study, the combination of 6.25mg/kg SM09419 with azacitidine (0.8 mg/kg QD) and/or venetoclax (25 mg/kg QD) induced significant TGI (95-98% vs. vehicle, p<0.001) with tumor regression at Day 26. Azacitidine + venetoclax induced 79% TGI (p<0.001), but no tumor regression was observed. The triple combination induced tumor regression in all mice and complete regressions in 4/6 mice (67%); it had a greater effect on slowing tumor regrowth after treatment discontinuation vs. a single agent or doublet. SM09419 alone or in combination was well tolerated in these xenograft models based on body weight measurements. In summary, SM09419 potently inhibited SRSF6 phosphorylation and Wnt signaling pathway activity and induced apoptosis in FLT3-ITD cell lines. It also inhibited proliferation in cell lines and primary AML cells regardless of FLT3 status. The strong in vivo antitumor effects observed as combination treatment suggest that SM09419 combined with standard therapies may provide a clinical benefit by slowing or preventing relapse in AML with a marker of poor prognosis such as FLT3-ITD. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.