Ningshu Liu

BAY 1125976, a selective allosteric AKT1/2 inhibitor exhibits high efficacy on AKT signaling‐dependent tumor growth in mouse models

The PI3K‐AKT‐mTOR signaling cascade is activated in the majority of human cancers, and its activation also plays a key role in resistance to chemo and targeted therapeutics. In particular, in both breast and prostate cancer, increased AKT pathway activity is associated with cancer progression, treatment resistance and poor disease outcome. Here, we evaluated the activity of a novel allosteric AKT1/2 inhibitor, BAY 1125976, in biochemical, cellular mechanistic, functional and in vivo efficacy studies in a variety of tumor models. In in vitro kinase activity assays, BAY 1125976 potently and selectively inhibited the activity of full‐length AKT1 and AKT2 by binding into an allosteric binding pocket formed by kinase and PH domain. In accordance with this proposed allosteric binding mode, BAY 1125976 bound to inactive AKT1 and inhibited T308 phosphorylation by PDK1, while the activity of truncated AKT proteins lacking the pleckstrin homology domain was not inhibited. In vitro, BAY 1125976 inhibited cell proliferation in a broad panel of human cancer cell lines. Particularly high activity was observed in breast and prostate cancer cell lines expressing estrogen or androgen receptors. Furthermore, BAY 1125976 exhibited strong in vivo efficacy in both cell line and patient‐derived xenograft models such as the KPL4 breast cancer model (PIK3CAH1074R mutant), the MCF7 and HBCx‐2 breast cancer models and the AKTE17K mutant driven prostate cancer (LAPC‐4) and anal cancer (AXF 984) models. These findings indicate that BAY 1125976 is a potent and highly selective allosteric AKT1/2 inhibitor that targets tumors displaying PI3K/AKT/mTOR pathway activation, providing opportunities for the clinical development of new, effective treatments.

MOLECULAR MECHANISMS AND COMBINATION STRATEGIES WITH PI3K AND BTK INHIBITORS TO OVERCOME INTRINSIC AND ACQUIRED RESISTANCE IN PRECLINICAL MODELS OF ABC-DLBCL

The Brutons tyrosine kinase (Btk) inhibitor ibrutinib has demonstrated promising efficacy in a variety of hematologic malignancies. However, the precise mechanism of action of the drug remains to be fully elucidated. Tumor‐infiltrating macrophages presented in the tumor microenvironment have been shown to promote development and progression of B‐cell lymphomas through cross talk mediated by secreted cytokines and chemokines. Because Btk has been implicated inToll‐like receptor (TLR) signaling pathways that regulate macrophage activation and production of proinflammatory cytokines, we investigate the immunomodulatory effects of Btk inhibitor on macrophages. Our results demonstrate that Btk inhibition efficiently suppresses production of CXCL12, CXCL13, CCL19, and VEGF by macrophages. Furthermore, attenuated secretion of homeostatic chemokines from Btk inhibitor‐treated macrophages significantly compromise adhesion, invasion, and migration of lymphoid malignant cells and even those not driven by Btk expression. The supernatants from Btk inhibitor‐treated macrophages also impair the ability of endothelial cells to undergo angiogenic tube formation. Mechanistic analysis revealed that Btk inhibitors treatment downregulates secretion of homeostatic chemokines and cytokines through inactivation of Btk signaling and the downstream transcription factors, NF‐κB, STAT3, and AP‐1. Taken together, these results suggest that the encouraging therapeutic efficacy of Btk inhibitor may be due to both direct cytotoxic effects on malignant B cells and immunomodulatory effects on macrophages present in the tumor microenvironment. This novel mechanism of action suggests that, in addition to B‐cell lymphomas, Btk inhibitor may also have therapeutic value in lymphatic malignancies and solid tumors lacking Btk expression.

Abstract 154: The phosphatidylinositol-3-kinase (PI3K) inhibitor (i) copanlisib is active in preclinical models of B-cell lymphomas as single agent and in combination with conventional and targeted agents including venetoclax and palbociclib

Introduction Copanlisib (BAY 80 6946) is a highly selective pan class I PI3K-i with predominant inhibitory activity against PI3Kδ and PI3Kα, in clinical development as single agent and in combination for lymphoma patients. To address single agent antitumor activity in different lymphomas and to understand the molecular basis of resistance mechanisms for rational combination, we performed a screening of copanlisib as single agent and in combination with 15 other anticancer agents in 17 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL). Methods MCL (Jeko1, Rec1, JVM2, Granta519, Maver1, Mino1, SP-49, SP-53, UPN1, Z138), MZL (Karpas1718, VL51, SSK41, ESKOL, HAIR-M, HC-1) and CLL (MEC1) cell lines were exposed to increasing doses of copanlisib alone and in combination with other compounds using the fixed ratio set-up. Tested compounds included approved and experimental inhibitors of key regulatory pathways. Synergy was assessed via Chou-Talalay combination index (CI). Gene expression profiling (GEP) was done using Illumina Human HT12Expression BeadChips and GSEA (FDR Results Copanlisib showed antitumor activity in most cell lines (median IC50=22nM; 95%C.I.: 15-98). The other most active drugs were bortezomib (5nM; 5-7), romidepsin (34nM; 2-94), roniciclib (23nM; 18-29), panobinostat (161nM; 11-1263), MI2 (490nM; 224-1000). The remaining had median IC50s >500nM. Copanlisib-containing combinations often gave synergy/additive effects: copanlisib with venetoclax was beneficial in 16/17; with MI2 in 15; with palbociclib or ibrutinib in 14; with BAY 1125976 or panobinostat in 13; with lenalidomide or BAY 1238097 in 12; with rituximab in 11; with romidepsin in 10; with roniciclib in 9; with bortezomib in 8; with BAY 1143572 in 7; with bendamustine in 6; with ruxolitinib in 2. Combinations with venetoclax and with palbociclib were the most promising, achieving CI values Conclusion Copanlisib was active in MCL, MZL and CLL models. Combinations with BCL2-i venetoclax and CDK4/CDK6-i palbociclib were the most synergistic. Specific GEP features might predict lymphomas that could benefit from these regimens. Citation Format: Eugenio Gaudio, Ivo Kwee, Filippo Spriano, Chiara Tarantelli, Andrea Rinaldi, Thibaud Jourdan, Melanie Berthold, Alberto Arriibas, Anastasion Stathis, Davide Rossi, Ningshu Liu, Martin Lange, Oliver Politz, Emanuele Zucca, Francesco Bertoni. The phosphatidylinositol-3-kinase (PI3K) inhibitor (i) copanlisib is active in preclinical models of B-cell lymphomas as single agent and in combination with conventional and targeted agents including venetoclax and palbociclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 154. doi:10.1158/1538-7445.AM2017-154

COMBINATORIAL SCREENING OF THE PI3K INHIBITOR COPANLISIB IN T CELL LYMPHOMAS

toxicology study was conducted in cynomolgus monkeys (cynos). Methods: Biotinylated unconjugated Ab was used for immunohistochemistry (IHC). Flow cytometry was used for measuring CD74 expression. STRO‐001 was used to determine the EC50 and percent span of killing in NHL cell lines. The anti‐tumor activity of STRO‐001 in NHL xenografts was examined. STRO‐001 was administered to cynos at doses of 1, 3, 10 and 30 mg/kg on days 1 and 15. Results: CD74 expression was evaluated by IHC on duplicate core (matched pair) biopsies. Medium to high CD74 expression in >70% of cells was observed in 86/100 DLBCL, 22/28 follicular lymphoma and 49/78 MCL samples. In vitro cytotoxicity assays show potent activity of STRO‐001 in a diverse panel of B‐cell tumor lines with EC50 values ranging from 0.17–13 nM. CD74 cell surface expression is required for STRO‐001 cytotoxicity, but expression level does not correlate with in vitro potency (R = 0.4154). STRO‐001 exhibits dose‐dependent tumor growth inhibition in rituximab‐resistant SU‐DHL‐6 xenografts starting at 2.5 mg/kg weekly × 3 doses. STRO‐001 + bendamustine/ rituximab (BR) further improves tumor suppression in SU‐DHL‐6 xenografts compared to vehicle (p = 0.002) or BR alone (p = 0.02). Studies with an MCL model, Jeko‐1, demonstrate robust STRO‐001 anti‐tumor activity compared to vehicle (p < 0.0001) starting at a single 3 mg/kg dose, with a single 10 mg/kg dose resulting in tumor regression for up to 64 days posttreatment. STRO‐001 treatment 14 days post tumor inoculation was used to evaluate disease progression in the disseminated Mino MCL model. Vehicle‐treated animals developed advanced progressive disease, with median survival of 81 days. In contrast, all Mino xenografts treated with STRO‐001 at 3 or 10 mg/kg were alive and disease free at the time of sacrifice 135 days post inoculation. STRO‐001 demonstrated B‐cell depletion in cynos, confirming the intended PD effect. Myelosuppression was observed at the highest dose, with no off‐target toxicity. Conclusions: STRO‐001 demonstrates potent in vitro cytotoxicity in NHL cell lines and anti‐tumor activity in NHL xenograft models, including prolonged survival in the disseminated Mino MCL model. STRO‐ 001 depletes B cells in a dose‐dependent manner. Clinical studies of this novel ADC for treatment of B‐cell malignancies are under development.

COPANLISIB IN COMBINATION WITH ANTI‐PD‐1 INDUCES REGRESSION IN ANIMAL TUMOR MODELS INSENSITIVE OR RESISTANT TO THE MONOTHERAPIES OF PI3K AND CHECKPOINT INHIBITORS

KARPAS299, KI‐JK), PTCL (FEPD, HH) and SS (H9, HUT78) were exposed to increasing doses of copanlisib alone and in combination with increasing doses of other compounds using the fixed ratio set‐ up. Tested compounds were anti CD30 antibody‐drug conjugate brentuximab, ALK‐i crizotinib, CDK‐i roniciclib, DNA damage agent bendamustine, HDAC‐i panobinostat and romidepsin, immunomodulatory lenalidomide, JAK1/2‐i ruxolitinib, BTK‐i ibrutinib, MALT‐i MI2, proteasome‐i bortezomib, BCL2‐i venetoclax, CDK4/6‐i palbociclib, the BET‐i BAY 1238097, and the PTEFb/CDK9‐i BAY 1143572. Synergy was assessed with Chou‐Talalay combination index (CI): synergism (<0.9), additive (0.9–1.1), antagonism/no benefit (>1.1). Gene expression profiling was done using the Illumina‐HumanHT‐12 Expression‐BeadChips and GSEA (FDR < 0.25). Results: Copanlisib had a median IC50 of 285 nM (50–1660 nM). Among the other compounds, the most active were bortezomib (IC50 3.1 nM; 1.6–6 nM), romidepsin (IC50 2.4 nM; 1.8–7.7 nM), panobinostat (IC50 10.2 nM, 3.8–14 nM), roniciclib (IC50 21.1 nM, 13.4–50.1 nM). Different copanlisib‐containing combinations, tested in the 9 cell lines, were synergistic: copanlisib with palbociclib (7/9 cell lines), panobinostat (7/9), BAY 1238097 (6/9), venetoclax (5/9), romidepsin (5/9), ruxolitinib (4/9), lenalidomide or BAY 1143572 or brentuximab or crizotinib (3/9). The most promising combinations were copanlisib/venetoclax and copanlisib/palbociclib, with a median CI < 0.7 in 3 cell lines. High expression of genes involved in interferon signaling and TP53 pathway were associated with synergism to copanlisib/venetoclax, while MYC target genes and cell cycle signaling were associated with resistance to the combination. Largely, the opposite was observed for copanlisib/palbociclib, with synergism in cells with high expression of E2F targets and genes involved in cell cycle and resistant in cells with expression of transcripts involved in interferon and TP53 signaling. Conclusion: Copanlisib was active in T‐cell lines derived from ALCL, PTCL and SS. The combinations with the BCL2‐inhibitor venetoclax and with the CDK4/CDK6‐inhibitor were the most synergistic and the specific GEP features might predict lymphomas that could benefit from these regimens.

Abstract 1045: Proteomic analysis of effects of MEK inhibition with BAY86-9766 on LKB1/AMPK and mTOR pathway in lung cancer cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: MEK inhibitors such as BAY86-9766 are a new class of agents that show promise in the treatment of non-small cell lung cancer (NSCLC). The downstream effects of MEK inhibition in NSCLC have not been fully elucidated. We performed a broad proteomic analysis to determine which signaling pathways were modulated by BAY86-9766 treatment and how these pathways correlate with sensitivity. Methods: We treated 109 lung cancer cell lines with MEK inhibitor BAY86-9766 at a concentration of 2200 nM. Drug sensitivity was determined by CellTiter-Glo assay and cell lines were classified as sensitive or resistant based on whether their IC50 values were in the highest or lowest 1/3rd of those tested. Using paired t-tests, we compared pre- versus post-treatment protein levels in the overall group and between the sensitive vs. resistant cell lines. Results: MEK inhibitor BAY86-9766 was effective in reducing phosphorylation of direct downstream signaling molecules pMAPK (p<0.0001) and p-P90RSK (p<0.02). There was no significant difference in the level of pMAPK suppression between sensitive and resistant cell lines (p=0.55). In contrast, LKB1/AMPK activation was observed following MEK inhibitor treatment, as illustrated by higher post-treatment levels of LKB1 total protein, pAMPK, and pTSC2 (all p values <0.02). As a consequence of these changes, the activity of downstream mTOR was suppressed, as evidenced by decreased pS6 (S235/236), pS6 (S240/244) and phospho-p70S6K and increased pPDK1 (all p≤0.002). All of these changes were significantly more pronounced in sensitive cell lines vs resistant cell lines (all p<0.01). We also saw evidence of activation of feedback loops, as Src significantly increased in sensitive cell lines compared to resistant cell lines (p=0.0009). Conclusions: We have performed broad proteomic analysis on cell lines treated with MEK inhibitor BAY86-9766 to determine which pathways are modulated by treatment and how they might relate to sensitivity. We conclude that MEK inhibition with BAY86-9766 may exert some of its effects by suppressing mTOR activity via the LKB1/AMPK pathway, and that the degree of modulation of the AMPK pathway correlates with sensitivity. This mechanism may involve decreased activation of p90RSK by MAPK, which leads to decreased degradation and increased activity of LKB1, and thereby increased AMPK activity and decreased mTOR signaling. This works suggests a rational basis for combinations of targeted agents to overcome resistance, such as combinations of MEK inhibitors with mTOR inhibitors or PI3 kinase inhibitors. Citation Format: Kathryn A. Gold, Lauren A. Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai T. Tran, Kevin R. Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis of effects of MEK inhibition with BAY86-9766 on LKB1/AMPK and mTOR pathway in lung cancer cell lines. [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 1045. doi:10.1158/1538-7445.AM2013-1045