ALBERT

Description: Tumor cells rewire metabolic pathways to meet the high metabolic demands of proliferation, frequently developing auxotrophy to specific amino acid(s) (AAs) required to satisfy protein biosynthesis. Thus specific metabolic inhibitors or AA-depleting enzymes have been developed and tested as cancer therapeutics. For example, depletion of asparagine by bacterial L-asparaginase (ASNase) has proven efficacious against hematologic malignancies, especially leukemia and lymphoma, by starving tumors lacking asparagine synthetase (ASNS). We and others have reported that the glutaminase (GLS) activity of ASNase is required for anticancer activity against ASNS-positive leukemia cell types in vitro.1 In vivo, we have found that durable response to ASNase in pre-clinical models of leukemia also requires glutaminase activity, even against ASNS-negative leukemia models; a glutaminase-deficient mutant of ASNase yielded subsequent leukemia recurrence. We speculate that the underlying anti-leukemia mechanism mediated by ASNase glutaminase activity involves a deeper depletion of asparagine within the tumor microenvironment, since ASNS in nearby cells (adipocytes, mesenchymal stromal cells, etc.) can use glutamine as a precursor for asparagine synthesis. Nevertheless, since L-glutamine depletion is thought to cause the significant side effects of ASNase, enzyme variants with reduced glutaminase coactivity are being developed and tested. Another viable therapeutic strategy involving glutamine starvation via GLS inhibitor has shown significant pre-clinical activity in acute myeloid leukemia (AML) and multiple myeloma (MM) models; this approach is synergistic with hypomethylating agents and BCL2 inhibitors in AML, and with proteasome inhibitors in MM. Recent findings highlight the switch to glutamine metabolism as a metabolic dependency of tyrosine kinase-driven AML, and targeting GLS in conjunction with tyrosine kinase inhibition has been proposed.2 Targeting arginine metabolism has been shown to be another viable therapeutic strategy. Arginine (ARG) depletion using pegylated arginine deaminase (ADI-PEG 20) or pegylated arginase (PEG-ARGase), the 2 critical enzymes of the ARG metabolism/urea cycle, reduced leukemia tumor burden in AML models characterized by low arginosuccinate synthetase (ASS) and high uptake of ARG. However, recently reported Phase I/II clinical trials of recombinant PEG-arginase and of ADI-PEG 20 showed minimal efficacy in relapsed/refractory AML and in solid tumors despite efficient depletion of arginine and low ASS1 expression in tumors, indicating that depletion of arginine alone is insufficient for clinical activity. As a final example of AA metabolic pathways targeted in the treatment of hematologic malignancies, exogenous L-cysteine is required for the synthesis of glutathione for antioxidant cellular defense. In pre-clinical studies, multiple malignancy subtypes were sensitive to cysteine and cystine degradation by an engineered human cyst(e)inase enzyme, including AML, acute lymphocytic leukemia, poor-risk chronic lymphocytic leukemia (CLL), and MM.3 In all therapeutic strategies targeting AA metabolism, the tumor microenvironment may contribute to resistance. For example, bone marrow stromal cells efficiently import cystine, convert it to cysteine, and transport it to CLL cells, facilitating leukemia chemoresistance. Mesenchymal stromal cells and bone marrow adipocytes secrete asparagine and glutamine, respectively, and protect leukemia cells from ASNase cytotoxicity. Recent insights into the immune tumor microenvironment highlight interplay between tumor, AAs, and immune cell functions. Some AAs, such as arginine and glutamine, are essential nutrients for immune cell proliferation and metabolism; excessive tumor consumption of glutamine, or secretion of arginase by myeloid-derived suppressor cells or AML blasts, may deprive immune cells, impair T cell proliferation, and induce immunosuppressive phenotypes. GLS inhibitors that block glutamine consumption and arginase inhibitors that increase plasma arginine, increase availability of their respective target nutrients for immune cells and are, therefore, being explored in ongoing clinical trials as monotherapies and in combination with anti-PD1 blockade. Chan WK, Lorenzi PL, Anishkin A, et al. The glutaminase activity of L-asparaginase is not required for anticancer activity against ASNS-negative cells. Blood. 2014;123:3596-3606. Gallipoli P, Giotopoulos G, Tzelepis K, et al. Glutaminolysis is a metabolic dependency in FLT3(ITD) acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition. Blood. 2018;131:1639-1653. Zhang W, Trachootham D, Liu J, et al. Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia. Nat Cell Biol. 2012;14:276-286. Disclosures Konopleva: Stemline Therapeutics: Research Funding. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties.

Description: T-ALL is an aggressive hematologic malignancy arising from immature T-cell precursors. Previous studies identified dependence of T-ALL (with a notable exception of early T-cell precursor (ETP) ALL) on BCL-XL (Chonghaile, Cancer Discovery 2014; Khaw, Blood 2016). However, BCL-XL specific inhibitors exhibit on-target toxicity of thrombocytopenia, restricting the use in acute leukemias (Vogler, Blood 2011). DT2216, a novel BCL-XL specific proteolysis targeting chimera (PROTAC), targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase, leading to BCL-XL ubiquitination and degradation selectively in cells express VHL (Khan, ASH 2018). Platelets lack VHL expression and therefore are spared from destruction by DT2216. Here we studied the pre-clinical efficacy of DT2216 in T-ALL cell lines in vitroand in vivousing T-ALL patient-derived xenograft (PDX) models. We first analyzed anti-apoptotic proteins (BCL-XL, BCL-2, MCL-1) expression in 4 B-ALL (LAZX2, MUTZ5, RS4:11, BALL1) and 6 T-ALL cell lines (SUPT1, KOPT1, Loucy, CCRF-CEM, PF384, Jurkat) by immunoblotting. This analysis demonstrated that ALL cell lines generally co-express BCL-XL and BCL-2 (Figure 1A). To identify functional dependencies, we utilized BH3 profiling that measures cytochrome C release after priming cells with BH3 peptides selectively targeting pro-survival BCL-2 family proteins in 4 B-ALL and 3 T-ALL cell lines. Similarly, cells were co-dependent on several anti-apoptotic members as shown by higher cytochrome c release in response to BIM, BID and BMF peptides targeting multiple anti-apoptotic proteins, and lower response to FS-1, ABT-199, HRK, MS-1, targeting individual anti-apoptotic members (Figure 1B). Analysis of the 3 B-ALL and 3 T-ALL PDX lines identified similar patterns that ALL cells are co-dependent on several anti-apoptotic members. Notably, we observed high cytochrome C release in response to mBAD that targets BCL-2 and BCL-XL; in addition, two of the three T-ALL PDXs, but none B-ALL PDX, responded to BCL-XL specific peptide HRK and to DT2216 confirming a functional role of BCL-XL in T-ALL survival. Next, we studied the sensitivity of ALL cells to ABT-199, DT2216 and the combination, in comparison with dual BCL-2/BCL-XL inhibitor ABT-263. DT2216 treatment (24hrs) caused a dose-dependent reduction of cellular viability in all 6 T -ALL and 3 B-ALL lines (except for BALL1 with complex karyotype refractory to all agents) measured by Cell TiterGlo assay, with T-ALL cells demonstrating a log higher sensitivity compared to B-ALL. In contrast, 5 out of 6 T-ALL lines (all besides ETP line Loucy) had no response to ABT-199, while 3 B-ALL lines showed dose-dependent response. All lines except BALL1 responded to ABT-263 (Figure 1C). Notably, the combination of DT2216 with ABT-199 synergistically reduced cell viability, with average CI of 0.3 (range 0.1-0.7 in all lines besides BALL1) (Figure 1D). Immunoblotting of DT2216 treated cells confirmed dose-dependent, on-target BCL-XL degradation as early as 6 hrs (Figure 1E). We next tested the therapeutic efficacy of DT2216 alone or combined with chemotherapy in T-ALL PDX models. NSG mice were engrafted with T-ALL PDX CU76 and D115. After documenting bone marrow (BM) engraftment by flow cytometry in BM aspirates on Day 14 post cell injection, mice were randomized to receive vehicle, chemotherapy ("VDL", VCR 0.15mg/kg, Dexa 5mg/kg, L-ASP 1000U/kg, ip., qw), DT2216 (15mg/kg, ip., q4d) or their combination for 3 weeks. Mice tolerated DT2216 therapy well, with no platelet toxicity by whole blood count 24hrs post the first and last DT2216 dosing. DT2216 reduced leukemia burden, delayed leukemia progression (Fig 1G) and significantly extended mice survival in both models. VDL chemotherapy had no effect on ALL progression in CU76 model and showed efficacy similar to DT2216 in D115 model; of importance, VDL+ DT2216 combination resulted in significant extension of survival in both chemoresistant and chemosensitive models (Figure 1F). In summary, T-ALL cells are functionally dependent on BCL-XL for survival and are highly sensitive to DT2216, while B-ALL are largely BCL-2 dependent and respond to BCL-2 inhibitors such as ABT-199. DT2216 alone and in particular when combined with chemotherapy reduced leukemia burden and prolonged survival in T-ALL PDX models. This study suggests targeting BCL-XL by DT2216 represents highly effective and safe adjunct therapeutic modality in T-ALL. Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Zhang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Ghotbaldini:CPRIT Research Grant: Research Funding. Zheng:Dialectic Therapeutics: Equity Ownership, Other: Co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Zhou:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents. Konopleva:Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Genentech: Honoraria, Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Forty-Seven: Consultancy, Honoraria; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.