ALBERT

Description: Notch1-mutated T-ALL is an aggressive hematologic malignancy lacking targeted therapeutic options. Genomic alterations in Notch1-gene and its activated downstream pathways are associated with metabolic stress response and heightened glutamine (Gln) utilization to fuel oxidative phosphorylation (OxPhos) (Kishton at al., Cell Metabolism 2016, 23:649, Herranz at al., Nat Med, 2015, 21(10): 1182-1189). Hence, targeting NOTCH1-associated OxPhos and/or Gln dependency could constitute a plausible therapeutic strategy for T-ALL. In this study we examined metabolic vulnerabilities of NOTCH1-driven T-ALL and tested pre-clinical efficacy of novel mitochondrial complex I (OxPhosi) IACS-010759 and of glutaminase inhibitor CB-839 (GLSi) in T-ALL models including Notch1-mutated T-ALL cell lines, patient-derived xenograft (PDX) and primary T-ALL cells. We have previously reported and confirmed in this expanded study the anti-leukemia efficacy of IACS-010759 (EC50s 0.1-15 nM) (Molina at al., Nat Med, 2018, 24: 1036; Baran at al., Blood, 2018, 132:4020). Metabolic characterization demonstrated that OxPhosi caused striking dose-dependent decrease in basal and maximal oxygen consumption rate (OCR), ATP and NADH generation in T-ALL cell lines and primary T-ALL samples (p

Description: L-asparaginase (ASNase) is a standard component of treatment regimens used for acute lymphoblastic leukemia and is being tested against other cancer types, including acute myeloid leukemia, lymphoma, and pancreatic cancer. We and others have reported that the anticancer activity of ASNase requires the enzyme's glutaminase activity, but the underlying glutaminase-mediated mechanism(s) that lead to leukemia cell death are unknown. Glutamine, the most abundant amino acid in the blood, is known for pleiotropic roles in numerous biological pathways, including energy metabolism, redox metabolism, nucleotide anabolism, and amino acid anabolism. Many cancer cells have been found to reprogram their metabolic pathways to become highly dependent on glutamine for survival and proliferation. Glutaminase (GLS/GLS2)-mediated conversion of glutamine to glutamic acid provides the latter as a substrate for conversion to α‐ketoglutarate by transaminases or glutamate dehydrogenases (GLUD1/GLUD2) to fuel the TCA cycle. Consequently, targeting glutamine metabolism has become an attractive strategy for anticancer therapy. The enzyme asparagine synthetase (ASNS) mediates resistance to ASNase through synthesis of asparagine. ASNS is expressed in most cell types, and its expression is upregulated in response to a wide variety of cell stresses, including amino acid limitation and endoplasmic reticulum stress. We and others have shown that ASNS-positive leukemia cells capable of synthesizing asparagine de novo are less responsive than ASNS-negative leukemia cells to ASNase therapy (Chan et al., Blood, 2014). Moreover, ASNase resistance has been associated with elevated ASNS expression. In fact, we have shown that ASNS expression is a predictive marker of the in vitro response of leukemia cell lines and some solid tumor cell types to ASNase. The expression of ASNS in most cells in the body poses a serious challenge for successful therapy with ASNase; for example, production of asparagine by the liver and cells (e.g., mesenchymal stem cells and adipocytes) of the tumor microenvironment may contribute significantly to ASNase resistance in vivo. Here we used the high-glutaminase E. chrysanthemi ASNase (Erwinaze®), wild-type E. coli ASNase (ASNaseWT), and the glutaminase-deficient E. coli mutant, ASNaseQ59L, as models of high, medium-, and low-glutaminase, respectively, to explore ASNase glutaminase activity-mediated mechanisms of leukemia cell death. Unexpectedly, we found that increasing glutaminase activity caused an increase in the suppression of ASNS upregulation in vitro (Figure 1A). In NSG mice injected with luciferase-labeled Sup-B15 cells, single-agent ASNaseWT yielded a durable response approximating cure, whereas glutaminase-deficient ASNaseQ59L yielded a complete response but with recurrence. Together, the results suggest that ASNase glutaminase activity is associated with suppression of ASNS upregulation, making durable, single-agent anticancer activity easier to achieve. Overall, the results provide new insight into the mechanism of action of ASNase. Disclosures Konopleva: Stemline Therapeutics: Research Funding. Weinstein:NIH: Patents & Royalties: L-asparaginase. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties.

Description: The expression of Bcl-2 family proteins is perturbed in multiple types of cancers, including leukemias, and is associated with disease progression and resistance to chemotherapy. ABT-199 (GDC-0199) is a new BH3 mimetic that was developed to specifically target Bcl-2 while sparing Bcl-XL, hence avoiding thrombocytopenia intrinsic to 1st generation BH3 mimetics like ABT-737 (Souers et al., Nat Med, 2013). In this study, we report proteomic profiling of Bcl-2 family members in a large series of ALL patients (pts) and pre-clinical activity of ABT-199. Expression of 20 pro- and anti-apoptotic proteins was studied in 186 newly diagnosed ALL using reverse phase protein arrays (RPPA). Supervised clustering demonstrated distinct differences in 11 proteins in ALL with different cytogenetic and FAB characteristics (Fig. 1, p4µM). Next, the cytotoxic activity of ABT-199 was tested against a panel of 12 genetically diverse primary ALL samples, including 6 from pts with relapsed or refractory disease. Ten out of twelve samples (83%) were exquisitely sensitive to both agents, with IC50 values of 0.0001-0.14µM for ABT-199 and 0.0004-0.3µM for ABT-737. One of the four Ph+ samples was resistant to both agents, and one of the two T-ALL was less sensitive to ABT-199 compared to ABT-737. Two samples with t(4;11) were highly sensitive to ABT-199. All primary ALL samples tested (n=7) expressed high levels of Bcl-2, and no significant correlation between sensitivity and expression of Bcl-2 family members was found. Importantly, three human-derived xenografts from pediatric pre-B-ALL samples (1345, 1809, 0398) were very sensitive to ABT-199 (IC50 3nM, 0.1nM and 2.3nM, respectively). Finally, anti-leukemia activity of ABT-199 was tested in MLL-rearranged patient-derived xenograft NSG mice. Treatment with ABT-199 at 100mg/kg/d by oral gavage days 13-23 significantly reduced leukemia tumor burden as determined by bioluminescence imaging (average 70% reduction in BLI signal in 4 ABT-treated mice compared to 4 control animals at 9 weeks, p=0.03). In summary, proteomic profiling and patterns of sensitivity to Bcl-2 inhibition indicate that ALL, with exception of Burkitt's lymphoma, represents a Bcl-2 dependent disease. These results provide strong rationale for introducing ABT-199, which recently showed impressive efficacy in CLL trials, into the clinical armamentarium of ALL therapy. Disclosures: Konopleva: AbbVie, Inc: Research Funding. Leverson:AbbVie, Inc.: Employment, Equity Ownership.

Description: Adult T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy characterized by limited therapeutic options and a high rate of treatment failure due to chemoresistance. T-ALL is largely driven by activating NOTCH1 mutations, where oncogenic NOTCH1 facilitates glutamine oxidation, induces metabolic stress, and facilitates reliance on oxidative phosphorylation (OXPHOS)1. In other malignancies, the shift toward OXPHOS-dependent high-energy status is associated with acquired chemoresistance. In this study, we found that the novel inhibitor of mitochondrial complex I (OXPHOSi) IACS-0107592 has preclinical activity in NOTCH1-mutated T-ALL; we also characterize the cellular and metabolic responses to OXPHOS inhibition and propose that an OXPHOSi be incorporated into standard-of-care therapy to improve outcomes in patients harboring NOTCH1-mutated T-ALL. Exposure to IACS-010759 (0-370 nM) in vitro drastically reduced T-ALL viability, with EC50 ranging from 0.1-10 nM for cell lines (n=7) and from 13-60 nM for patient-derived xenograft (PDX)-derived and primary T-ALL cells (n=10) (Fig.1). Oral administration of IACS-010759 (7.5 mg/kg/day) significantly reduced leukemia burden and extended overall survival (p

Description: Evasion of apoptosis is a key hallmark of cancer. BCL-2 family proteins, the central regulators of apoptosis, are often aberrantly expressed in tumors. Pro-apoptotic BCL-2 members bind and sequester anti-apoptotic BCL-2 proteins via their BH3 domains. Thus, BH3 mimetics represent a promising direction in cancer drug development. ABT-263, designed as a BH3 mimetic to inhibit BCL-2, BCL-XL, and BCL-W, has demonstrated efficacy in preclinical and clinical studies. However, thrombocytopenia is common in patients treated with ABT-263 due to the inhibition of BCL-XL, which is indispensable for platelet survival. ABT-199 (GDC-0199), a second-generation BH3 mimetic, has higher affinities for BCL-2 protein (Ki 〈 0.01 nM), which enhances the specificity of this agent to kill cancer cells without provoking unwanted thrombocytopenia (Souers, et al, Nature Med, 2013). Since BCL-2 is often overexpressed in hematological malignancies including acute myeloid leukemia (AML), we evaluated the anti-cancer effects of ABT-199 on AML cells. As a measure of BCL-2 specificity, BCL-XL overexpression in sensitive HL-60 cells resulted in complete resistance to ABT-199, while BCL-2 overexpression in these cells conferred moderate resistance to apoptosis induction. Moreover, OCI-AML3 cells with high MCL-1 levels were highly resistant to ABT-199, while knockdown of this protein greatly sensitized cells to this BH3 mimetic. Among 12 genetically diverse AML cell lines tested, seven were sensitive to ABT-199-induced apoptosis with 48-h EC50 ranging from 1.5 nM to 145 nM. In these seven sensitive, BCL-2 dependent cell lines, ABT-199 was uniformly more potent than ABT-737 (another BCL-2 inhibitor with a spectrum similar to ABT-263, p = 0.016). Next, we tested ABT-199 in 15 primary samples from relapsed/refractory AML patients. Twelve patient samples showed high sensitivity to apoptosis induction following 48-h exposure to ABT-199 (EC50 〈 10 nM). In a larger set of 23 cryopreserved AML patient samples, including AML cells with diploid cytogenetics and mutations in FLT3, NRAS, and NPM1 genes, 18 (78%) were sensitive to ABT-199 (100 nM). However, samples from patients with complex cytogenetics, t(8;21) and JAK2 mutation (n = 12) were largely insensitive to ABT-199 (17% response rate). Interestingly, in five of six primary AML samples with high blast counts, ABT-199 induced marked apoptosis in CD34+/CD38- AML stem/progenitor cells compared to bulk AML blasts (p = 0.01). Quantitative Western blot was used to determine BCL-2 protein levels in AML cell lines. Spearman analysis showed that EC50 of ABT-199 correlated negatively with BCL-2 protein expression (r = -0.605, p = 0.0143) and correlated positively with BCL-XL protein expression (r = 0.633, p = 0.0101). Similar correlations were also observed in primary AML samples, suggesting that pre-treatment cellular BCL-2 and BCL-XL levels might have utility as predictive markers of ABT-199 sensitivity. We next examined the in vivo anti-leukemic efficacy of ABT-199 in NOD SCID gamma (NSG) mice injected with luciferase-labeled MOLM-13 cells. The mice were treated with ABT-199 by daily oral gavage (a 2-wk treatment at dose of 100 mg/kg). Bioluminescence imaging showed that ABT-199 treatment significantly inhibited leukemia burden, which was also manifested by smaller spleen size and prolonged overall survival (p = 0.0004) when compared to the vehicle-treated mice. Furthermore, a 2-wk ABT-199 treatment significantly reduced leukemia burden (〉 50%) in bone marrows of NSG mice engrafted with primary FLT3-mutated AML cells (i.e., a mean of 70 ± 16% human CD45+ cells in bone marrow of control mice (n = 9) versus 32.7 ± 12% in ABT-treated mice (n = 11), p = 0.00002). Conclusions: the in vitro and in vivo efficacy data indicates that ABT-199 is a selective BCL-2 inhibitor, a potent apoptogenic agent, and hence a promising candidate for AML BCL-2-targeted therapy. Disclosures: Leverson: AbbVie, Inc.: Employment, Equity Ownership. Konopleva:AbbVie: Research Funding.

Description: We and others have previously reported that leukemia progression is associated with vast expansion of the hypoxic niches and stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in leukemic cells (Frolova et al. Cancer Biol Ther. 2012, 10:858; Benito et al. PLoS One 2011, 6(8); e23108:1). Interactions of leukemia and the bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells, and we have shown that HIF-1α stabilization in stromal cells of the microenvironment facilitates leukemia homing and progression (Chen et al. Blood 2012, 119:4971). In this study, we aimed to characterize the time-dependent progression of BM hypoxia involving both leukemia cells and components of the BM niche, using the multiphoton intravital microscopy (MP-IVM) technique. We first generated a transplantable, imageable leukemia model by retrovirally transducing C57Bl6-Ai14 murine BM cells that express red fluorescing tdTomato with the p190-Bcr/Abl oncogene. The resulting p190-Bcr/Abl tdTomato cells caused rapid development of acute lymphocytic leukemia (ALL) in un-irradiated C57Bl6 immunocompetent mice, manifested by infiltration of the spleen, liver, BM within long bones, skull, and central nervous system followed by death within 28 days. Leukemia cells collected from the BM (LBC) of these animals were transplantable into secondary recipients and triggered accelerated ALL development (14-16 days). Time-course analysis of skull and femur bones in the secondary recipients by MP-IVM demonstrated LBC lodging on day 1 after ALL cell injection, followed by rapid accumulation of leukemia cells localized predominantly within the sinusoidal spaces, which were visualized by injecting the vascular fluorescent dye BSA-647 (Fig. 1a). To detect in vivo hypoxia development, we utilized HS680 (HypoxiSense 680), a carbonic anhydrase IX (CAIX)–targeted fluorescent agent that can be used to image overexpression of CAIX, a direct HIF-1α target, in tumors in response to regional hypoxia. C57Bl6 mice were engrafted with 2 x 105 LBC , and HS680 was injected intravenously at serial intervals followed by MP-IVM. In two separate experiments, increased HS680 fluorescence was detected in bone-lining cells in the BM niches of mice harboring ALL on days 8 and 13, but not in their healthy littermates (Fig 1b). To obtain an independent confirmation of hypoxia, additional mice (n=3) at the same stage (day 14) of leukemia development were sacrificed 3 hr after injection of chemical hypoxia probe pimonidazole (Pimo), and hypoxic BM cells that bound the hypoxia probe were detected by immunohistochemistry. Pimo staining demonstrated vastly spread areas of hypoxia that enclosed both leukemia cells and BM niche cells (Fig 1c), consistent with our previously published observations in different leukemia models. In summary, these findings demonstrate rapid development of intra-BM hypoxia that parallels leukemia progression and involves not only leukemia cells, but also BM niche cells. The HS680 probe can detect hypoxia in vivo within niche cells but not in leukemia cells, likely because of differential expression of CAIX. Our ongoing studies will characterize the cellular origin of hypoxic niche cells by utilizing immunohistochemical techniques and Col2.3-GFPemd transgenic mice to visualize osteoblasts. We postulate that the tumor microenvironment altered with hypoxic niche cells will influence leukemia development or responses to therapy. To this end, we have generated mice with conditionally deleted HIF-1α within BM stromal cells and are investigating the differences in leukemia homing, progression, and chemoresistance between these mice and mice whose BM stromal cells express HIF-1a. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Studies have previously described the feasibility of receptor-mediated protein transfer in a cell culture model of Fanconi anemia (FA) group C. This study explores the versatility of this approach by using an antibody single-chain fusion protein to correct the phenotypic defect in FA group F cells. A 68.5-kd chimeric protein (His-M195FANCF) was expressed, consisting of a His tag, a single-chain antibody to the myeloid antigen CD33, and the FANCF protein, as well as a 43-kd His-FANCF fusion protein lacking the antibody motif, inEscherichia coli. The nickel-agarose–purified His-M195FANCF protein bound specifically to the surface of HeLa cells transfected with CD33 and internalized through vesicular structures. The fusion protein, but not CD33, sorted to the nucleus, consistent with the known nuclear localization of FANCF. No similar binding or internalization was observed with His-FANCF. Pretreatment of the transfected cells with chloroquine abolished nuclear accumulation, but there was little change with brefeldin A, indicating a minimal if any role for the Golgi apparatus in mediating transport from endosomes to the cytosol and the nucleus. The intracellular half-life of His-M195FANCF was approximately 160 minutes. Treatment of CD33-transfected FA group F lymphoblastoid cells with 0.1 mg/mL His-M195FANCF conferred resistance to mitomycin C. No similar protection was noted in CD33− parental cells or CD33+ FA cells belonging to groups A and C. These results demonstrate that antibody-directed, receptor-mediated protein transfer is a versatile method for the delivery of biologically active proteins into hematopoietic cells.

Description: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic malignancy of plasmacytoid dendritic precursor cells. Lacking standard therapy due to limited understanding of biology and historical rarity of the disease, clinical outcomes for BPDCN patients remain poor. Surface overexpression of CD123/IL3Rα is seen 70-80% of patients with AML (Han et.al, Clin Cancer Res, 2017) and in nearly 100% of BPDCN cases, with a markedly high intensity compared to normal hematopoietic stem cells, thus making CD123 an attractive target for BPDCN treatment. IMGN632 is a CD123-targeting antibody-drug conjugate (ADC) comprised of a novel humanized anti-CD123 antibody G4723A linked to a DNA mono-alkylating payload of the indolindobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. IMGN632 demonstrates potent activity in AML samples at low concentrations with minimal impact on normal bone marrow progenitors, and anti-leukemia effects in AML xenograft models (Kovtun et.al., Blood Advances, 2018). A phase I clinical trial of IMGN632 in relapse/ refractory AML (NCT033865) is now being performed and achieved encouraging results. We thus explored the anti-tumor effect of IMGN632 in BPDCN. We first tested the in vitro activity of IMGN632 in a BPDCN cell line CAL-1. Cells were exposed to control antibody or IMGN632 at various concentration for 96 hours, after which viable cell numbers were measured using AnnexinV /DAPI assay with counting beads by flow cytometry. IMGN632 demonstrated cytotoxic activity at 0.2μg/ml, with stronger effects at concentrations of 2μg/ml and above (Figure 1A). Next, the in vivo efficacy of IMGN632 was evaluated in BPDCN patient-derived-xenograft (PDX) models. NSG mice (6-8 weeks old) were injected with 1e6/mouse BPDCN cells by tail vein 24 hours after 250cGY sublethal irradiation. Upon confirmation of the bone marrow engraftment of hCD45/hCD123 cells by bone marrow aspiration, mice were randomized to 5 or 8 mice/group and received weekly intravenous vehicle, control antibody or IMGN632 by tail vein injection 24 hours following FcR blockade (400mg/kg naked antibody by intraperitoneal injection) for 3 weeks. Bone marrow engraftment and survival were monitored. For model A, the mice received either 24μg/kg or 240μg/kg of IMGN632. For model B, the mice received 240μg/kg IMGN632. Both samples responded to the IMGN632 treatment, with the engraftment lower than 1% in the bone marrow after 3 doses, compared with over 90% in vehicle and control antibody group (Figure 1B). All mice from vehicle and control antibody groups in model A died by 52 days after cell injection; the 24μg/kg IMGN632 treated mice had prolonged survival with median survival of 111 days (p

Description: Metabolic reprogramming of the key energy-generating pathways has been long recognized as one of the key oncogenic properties of cancer including leukemia. While accelerated glycolysis is considered to be most common feature of tumors, reliance on oxidative phosphorylation (Oxphos) as a major energy source has been reported for various tumor types. IACS-010759 is a novel OxPhos inhibitor(OxPhosi) that blocks cellular respiration through inhibition of complex I (Molina et al., AACR2016 Abstract #335) and considered as validated drug with clinical relevance in AML and solid tumors. Treatment of adult T-ALL remains unsatisfactory, with approximately one-third of patients experience disease relapse, and novel treatment strategies are warranted. In this study, we report pre-clinical activity of IACS-010759 in T-ALL models and characterize a cellular metabolic profile of T-ALL. Analysis of a panel of T-ALL cell lines showed that IACS-010759 significantly reduced viability measured by CTG assay in all cell lines tested (Notch mutant: Pf382, 1301, Jurkat, MOLT-4, P12-Ichikawa and Notch wt: T-ALL1). T-ALL cells displayed high sensitivity pattern to OxPhos inhibition with EC50 between 0,001 and 10 nM at day 5 analyzed by CTG assay (Fig.1). This reduction of cell viability was primarily due to cell cycle arrest demonstrated by reduction in EdU uptake, and moderate induction of apoptosis in selected T-ALL cell lines. In primary T-ALL samples from patients with newly diagnosed or relapsed/refractory ALL (n=2), in vitro 5-day treatment with IACS-010759 reduced viable cell number at EC50 of 13 nM and 45 nM, respectively. In primary human T-ALL PDX xenografts study, daily oral administration of IACS-010759 at 7.5mg/kg/qd was well tolerated, caused significantly reduced circulating leukemia burden and extended median survival duration (Fig.2). The mitochondrial fuel usage that characterizes Oxphos dependency in T-ALL cell line PF382 was analyzed by Mito fuel Test using the Seahorse Bioscience XF96 Analyzer. Among all three energy sources, PF382 depends most on free fatty acids (FA), indicating strong coupling to Oxphos and TCA cycle (Fig.3). Treatment of T-ALL with IACS-010759 had effectively inhibited FA-stimulated mitochondrial respiration indicated by decreased oxygen consumption rates (OCR) (Fig.4A). However, the cells maintain an ability to generate energy via glycolysis, indicated by high extracellular acidification rate (ECAR) in both, control and IACS-treated groups (Fig.4B). Next, mitochondrial function of T-ALL cells (PF382, Jurkat, 1301, P12Ischikawa, MOLT4, TALL1) was investigated using Mito Stress Test in Seahorse Bioscience XF96 Analyzer. IACS-010759 exposure for 2 hrs caused a striking dose-dependent decrease in basal and maximal OCR, reduction of proton leak and ATP production (Fig.5A, B, C), confirmed by the decreased ATP/ADP and NADH/NAD ratios measured by luminescence assays (ADP/ATP Glow assay, NADH/NAD Glow assay), consistent with inhibition of Oxphos. Conclusions: Taken together, these data provide information about metabolic profiling of T-ALL and indicate that Oxphos inhibition constitutes a novel therapeutic approach that targets a unique metabolic vulnerability of T-ALL cells. Further preclinical evaluation of Oxphos inhibitors in T-ALL is warranted. Disclosures Jabbour: ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.