Primary Acute Myeloid Leukemia Cells Research Articles

DJ4 Targets Rho-associated Protein Kinase Pathway and Attenuates Disease Progression in Pre-clinical Murine Models of Acute Myeloid Leukemia

Introduction: The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the diseasemotivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for variousactin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not beingsuccessfully utilized clinically towards cancer treatment. ROCK 1 and 2 overexpression has been linked to AML celllines and overall survival of AML patients. This work reports the considerable therapeutic efficacy of the ROCKinhibitor DJ4 in both in vitro and in vivo preclinical models of AML to highlight the potential of this class of inhibitors.Experimental Design: The cytotoxic and pro-apoptotic activities of DJ4 for primary human AML samples and humanAML cell lines was determined by cell viability (MTS), colony forming assays, and Annexin V assays. Immunoblotanalysis was used to detect phosphorylation of downstream substrates of ROCK. To assess the preclinical therapeuticefficacy, the luciferase-expressing human AML cell line OCIAML-3-YFP-Luc was injected subcutaneously (SC) andintravenously (IV) into NRG-S mice. Mice were treated through an intraperitoneal (IP) injection with either vehiclecontrol or DJ4 (10 mg/kg) for 3 weeks. Modified AML cell lines, OCI-AML3-YFP-Luc and MV4-11-Luc2-EGFP, werealso treated with their respective IC50 dose of DJ4 or with vehicle control for 24 h and then administered via IV intoNRG-S mice and the survival advantage was monitored over time without further treatment. Disease progression wastracked in mice studies by flow cytometry analysis and examining the survival, bioluminescent signal, tumor volume,and tumor weights of the animals over time.Results: DJ4 induced cytotoxic and pro-apoptotic effects, within the micromolar range and in a dose-dependentmanner, in human AML cell lines (IC50: 0.05-1.68 μM) and primary patient cells (IC50: 0.264-13.43 μM) that harboredvarious mutations; however, normal hematopoietic cells are largely spared (Figures A-C). Treatment of DJ4demonstrated ~5-fold selectivity towards AML patient samples relative to the CB-MNCs of healthy donors (IC50= 25μM, Figure B). Representative flow cytometry plots of the Annexin V assay with AML primary cells (Figure C) depictedthe increase in the apoptotic populations as a result of treatment with increasing concentrations of DJ4. ROCKinhibition by DJ4 disrupts the phosphorylation of downstream targets, myosin light chain (MLC2), and myosin bindingsubunit of MLC phosphatase (MYPT) in OCI-AML3 and MV4-11, yielding a potent yet selective treatment response atmicromolar concentrations, 0.02 to 1 μM (Figure D). Analysis of mice treated with DJ4 via IP for 2.5 weeks indicatedthat DJ4 was well-tolerated with comparable CBC with differential and chemistry values and tissue morphology fromcross-sections of the spleen, liver, lung, and kidney relative to the vehicle treated group. Mice that were IV or SCinjected with OCIAML3-YFP-Luc and treated via IP with DJ4 exhibited an increase in overall survival and reductionin disease progression relative to the vehicle treated control mice (Figure E). Towards the end of the study, theOCIAML3-YFP-Luc SC injected mice exhibited at least a 3-fold reduction in the bioluminescent signal, ~4-fold decreasein tumor volume, and the tumor weight was significantly reduced by ~3-fold among the DJ4 treated group relative tothe control (Figure E). Mice that were IV administered DJ4 pretreated AML cells also exhibited a decreasedbioluminescent signal, increased survival of 10 and 20 days, and a significant reduction in the percent of human CD45cells in the bone marrow or spleen, indicating a diminished tumor burden (Figure F).Conclusion:The observed potency of DJ4 towards various AML cell lines and primary samples with a diverse set of mutationssuggests that this is a promising candidate to be incorporated into the standard AML regimen to help many differentsubsets of AML patients. Therapeutics that induce apoptosis have been shown to be promising candidates towardovercoming chemoresistance and to work well with appropriate combinations of standard of care drugs. This workhighlights the potential of targeting the Rho-ROCK pathway to improve the prognosis of AML and the need for thefuture development of chemotherapeutics that are both less toxic and more effective. [Display omitted] DisclosuresClaxton: Astellas: Other: Clinical Trial; Novartis: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Daiichi Sankyo: Research Funding; Incyte: Research Funding.

Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK Cell-Mediated Cytotoxicity Against Acute Myeloid Leukemia Cells

Background: The immune checkpoint molecule B7-H3 (CD276) is overexpressed in various solid tumors and hematological malignancies; however, its expression is limited in normal tissue, which makes it an attractive therapeutic target in cancer. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in acute myeloid leukemia (AML) remains unexplored. Here, we hypothesized that targeting B7-H3 using mAbs alters the immunomodulatory function of B7-H3 and enhances NK cell-mediated cytotoxicity against AML cells.Methods: B7-H3 protein expression was analyzed in the peripheral blood (PB) and bone marrow of 100 patients with AML and 20 healthy donors by flow cytometry and tested for associations with multiple clinical parameters and disease outcomes. To investigate B7-H3's role in immunomodulation, we stably knocked down B7-H3 in AML cell lines including OCI-AML3, MV4-11, and U937 and co-cultured them with activated human NK cells. NK cell-induced apoptosis was measured by annexin-v binding assay using an IncuCyte live-cell imaging system. B7-H3-blocking mAbs (clones T-1A5, HEK5-1B3, and 58B1) were tested for their effect on NK-cell-mediated cytotoxicity in AML cell lines using live-cell imaging. AML xenograft (OCI-AML3) or patient-derived xenograft (PDX) models were used to determine the effect of anti-B7-H3 antibodies on AML growth. In vivo AML growth was monitored by measuring human CD45 positive cells with flow cytometry. A human-mouse chimeric (ch) antibody was generated based on sequences from the T-1A5 antibody, and its binding site on the B7-H3 protein was characterized by epitope mapping. Further, we evaluated the effect of chT-1A5 on NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in primary AML cells and healthy donor PB-derived mononuclear cells (PBMCs).Results: Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p < 0.01) and was higher in CD34 positive than in CD34 negative AML cells (p < 0.01). High B7-H3 expression was associated with poor overall survival (p = 0.04) and prognostic risk scores (p = 0.05). NK cell-mediated apoptosis was 3-fold higher in all 3 B7-H3-knockdown AML cell lines than in scrambled control cells, suggesting that B7-H3 is an important immunomodulator of NK cells. Moreover, we observed a significant increase in NK cell-mediated killing of AML cells in the presence of anti-B7-H3 mAbs (p < 0.01). In vivo, anti-B7-H3 antibodies significantly inhibited AML growth and extended survival in PDX models compared to IgG control treatment. Among PDX-bearing mice treated with the three anti-B7-H3 antibodies, the T-1A5 antibody-treated group survived for longer than the other groups. In combination with NK cells, T-1A5 treatment also significantly increased the survival of AML xenograft-bearing mice compared to NK + IgG control treatment. These data suggest that the T-1A5 antibody blocks B7-H3 and enhances NK cell-mediated cytotoxicity in AML cells in vivo. Next, we found that a chT-1A5 antibody induced NK cell-mediated ADCC in primary AML cells and cell lines in a dose-dependent manner. In contrast, it did not induce any ADCC activity in healthy donor-derived PBMCs, suggesting that chT-1A5 is not toxic to healthy cells. Moreover, the chT-1A5 antibody combined with human NK cells dramatically inhibited leukemia growth and extended survival in B7-H3 positive AML PDX models (p < 0.001) compared to the control (rituximab-treated) group. Finally, epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region of B7-H3 as the binding site for the T-1A5 antibody, which may be involved in the immunomodulatory function of B7-H3.Conclusion: B7-H3 is overexpressed in AML cells, and its expression is associated with poor overall survival. Anti-B7-H3 antibodies block B7-H3's immunomodulatory function and inhibit AML growth in vivo. A chT-1A5 antibody in combination with NK cells induced ADCC in primary AML cells in vitro and in vivo but had no effect on PBMCs from healthy donors. Therefore, targeting B7-H3 could benefit AML patients, specifically those with a poor clinical prognosis. DisclosuresBattula: Tolero Pharmaceuticals: Research Funding.

Isoform Selective HDAC8 Inhibitor (OCH3) Shows Potency in Selectively Targeting Primary AML Cells

The treatment outcomes for patients diagnosed with acute myeloid leukemia (AML) are still dismal. Recent advances in understanding AML indicate that the lack of efficacy is primarily due to non-specificity of currently used chemotherapeutics targeting both leukemic stem/progenitor cells (LSC) and normal hematopoietic stem cells (HSC). Thus, a critical barrier is the identification of innovative therapies that selectively target LSC. Histone deacetylase 8 (HDAC8) has been shown to enhance p53 protein deacetylation, which results in inactivation of p53, promoting LSC survival. We hypothesize that enzymatic/non-enzymatic role of HDAC8 is critical for LSC survival but not for HSCs. Then, we characterized our two tetrahydroisoquinoline (TIQ)-based selective HDAC8 inhibitors (HDAC8i) BIP and OCH3 for growth inhibition, apoptosis, activation of caspase 3, integrity of mitochondrial membrane potential (MMP), and acetylation of histone H4 in human leukemia cell lines. The growth inhibitory effects observed in cell lines were validated using bone marrow (BM) or peripheral blood (PB) cells from AML patients. Colony forming cell (CFC) assays were performed using AML BM/PB cells treated with OCH3 or BIP. OCH3 and BIP were also tested for hematotoxicity using normal CB CD34+ cells. Furthermore, we compared class I HDAC isoform engagement in human normal cord blood (CB) CD34+ cells and in SET-2 leukemia cells using our novel photoreactive probe TH1143. In CD34+ cells, TH1143 had higher level of engagement for HDAC1 and 2, whereas engagement of HDAC3 and 8 was minimal. In SET-2 cells, HDAC3 and HDAC8 displayed relatively higher engagement with TH1143 indicating HDAC engagement is likely cell type specific.The biological efficacies of OCH3 at 50uM and BIP at 25uM were noted to exert >50% growth inhibition in KG1 and in K562 leukemia cells. Both OCH3 and BIP significantly increased the number of apoptotic cells and there was an enhanced active caspase-3 activity. Furthermore, OCH3 and BIP treated cells displayed lower red/green ratio in comparison to control, indicative of poor MMP and depolarization to induce apoptosis (Table 1.a). OCH3 and BIP were further validated by using BM/PB cells from AML patients showing growth inhibition. This was also accompanied by increase in apoptotic cells by OCH3 and BIP. In contrast to BIP, OCH3 spared CB CD34+ cells as demonstrated by notably lower growth inhibition, apoptotic cells vs control when compared with primary AML cells from patients. Both OCH3 and BIP displayed minimal inhibition of CFU growth in CD34+ cells. However, HDAC8i induced significant CFU growth inhibition in primary AML samples suggesting that HDAC8i spares normal CFU progenitors but not leukemia progenitors (Table 1.b). Notably, both BIP and OCH3 lack ability to exert acetylation of histone H4, unlike broad spectrum HDAC inhibitor TSA (MFI with OCH3=0.96±0.03, BIP=0.77±0, TSA =1.63±0.15) which is consistent with isoform selectivity of OCH3 and BIP.The leukemia growth inhibitory effects at LSC level was demonstrated using ex vivo OCH3 treated AML patient derived BM/PB cells transplantation in humanized immunodeficient NSGS mice. After 10 to 12 weeks of transplantation mice receiving untreated AML cells had 7.73±2.18% while with OCH3 treatment mice had 4.84±1.37% human CD34+ leukemia cells, a 38% reduction in CD34+ leukemia cells, despite only a single ex vivo exposure to OCH3. Furthermore, in a second model, NSGS humanized mice were transplanted (IV) with primary leukemia cells from AML patients and after 4 weeks injected (IP) with OCH3 or vehicle control. After 12 weeks of transplantation in this second model human primary AML cell burden was 5.74±1.31% (OCH3) and 18.13±12.76% (vehicle control), while mice transplanted with normal CD34+ cells treated similarly with OCH3 or vehicle control displayed no detectable inhibition of human myeloid cell chimerism (OCH3:12.28 ± 3.31% vs vehicle control: 17.92±11.96%).Taken together, our data indicate that HDAC8 isoform inhibitor, OCH3 displayed significant inhibition of primary AML patient derived leukemia cells growth in vitro and in vivo in contrast to normal CD34+ cells. Selective inhibition of HDAC8 is sufficient to cause growth inhibition in primary AML progenitors including LSCs in vivo while sparing normal HSCs thus offer opportunities for further development of HDAC8i as new experimental therapeutics in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IL-1 Via IRAK1/4 Sustains Acute Myeloid Leukemia Stem Cells Following Treatment and Relapse

Current treatment options for relapsed acute myeloid leukemia (AML) are limited and ineffective for the majority of patients. In AML, primitive leukemia stem cells (LSCs) and pre-leukemic populations are able to maintain the disease and drive relapse. Thus, therapies targeting LSC populations may increase the overall survival of AML patients. In this study, we aim to identify the drivers favoring LSC expansion following treatment and relapse and develop potential therapies for AML. The transcriptome analyses of 12 pairs of functionally defined LSC fractions at diagnosis and relapse revealed significant changes of IL-1 signaling in AML patients. We demonstrated that the protein expression levels of interleukin-1 receptor type I (IL1R1) and its complex member interleukin-1 receptor accessory protein (IL1RAP) were both up-regulated in human leukemia stem and progenitor cells (LSPCs) at diagnosis or in relapse compared to normal hematopoietic stem and progenitor cells (HSPCs). Knockdown of IL1R1 and IL1RAP suppressed the clonogenicity and engraftment growth of primary human AML cells but showed low impacts on HSPCs in the normal bone marrow. Additionally, knockout of IL1R1 in leukemia MLL-AF9 mice significantly reduced the LSC frequency and prolonged the overall survival rate. To target IL-1/TLR signaling in LSCs, we performed iterative structure-activity relationship (SAR) guided medicinal chemistry, in silico modeling and leukemia cell line reporter assays to screen and identify a novel interleukin-1 receptor-associated kinase 1/4 (IRAK1/4) inhibitor (termed UR241-2). UR241-2 robustly inhibits IL-1/TLR signaling in AML cells including the activation of NF-κB following IL-1 stimulation. UR241-2 repressed LSPC function as assessed by colony-forming unit assays in primary human AML cells at diagnosis and in relapse while minimally impacting normal HSPC function. Taken together, our findings demonstrate the important role of IL-1/TLR signaling in supporting AML LSC expansion following treatment and relapse and suggest that targeting IL-1/TLR signaling using the novel IRAK1/4 inhibitor, UR241-2, can target LSC function to improve patient outcomes in AML. DisclosuresNo relevant conflicts of interest to declare.

A Novel Lair-1 Antibody Selectively Targets Acute Myeloid Leukemia (AML) Stem Cells

Extensive research has led to recent approval of novel therapies such as mylotarg, venetoclax, glasdegib and CC486, and small molecule inhibitors against actionable mutations such as ivosidenib (IDH1), enasidenib (IDH2), gliteritinib and midostaurin (FLT3) in AML. However, the mainstay of treatment in AML remains unchanged since the 1970s. There is a significant unmet need for AML patients that fail to respond to or relapse after standard-of-care (SOC) treatments including allogeneic stem cell transplantation and targeting actionable mutations. In addition, a large fraction of SOC patients invariably relapse due to persistence of chemotherapy-resistant leukemia stem cells (LSCs) or immune evasion. Therefore, identification of unique therapies that preferentially target elusive LSCs and promote immune responses to AML to prevent relapse are highly sought after. Unlike, targeting acute lymphoblastic leukemia (ALL) with CD19 or CD22 with various modalities, when developing AML therapies, it is of paramount importance to differentiate LSCs from hematopoietic stem cells (HSCs) to lessen or abolish unavoidable cytopenias.Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an immune checkpoint receptor on T cells and myeloid cells that delimits immune cell activation through binding to endogenous collagen ligands. In addition, LAIR-1 is universally expressed on AML blasts and may sustain AML survival signals. We demonstrated using multi-color flow cytometry that LAIR-1 is highly expressed in AML blasts (n=9 of 9) and that LAIR-1 expression in LSCs (markers: CD34 +CD38 -CD90 -CD45RA +/- or CD34 -CD117 +CD244 +/-) is high compared with negligible expression of LAIR-1 in HSCs (markers: CD34 +CD38 -CD90 +CD99 -) (n=3) (Figure 1). Based on these findings, we hypothesized that a LAIR-1 monoclonal antibody (mAb) would disrupt LAIR-1 mediated survival signaling and preferentially target LAIR-1 expressing AML LSCs and blast cells but not HSCs. To test this, we developed a novel LAIR-1 targeting mAb with a functional human IgG 1 isotype that blocks LAIR-1 binding to its ligands (including collagens, complement component C1q, MBL and SP-D)To characterize the anti-leukemic effect of the LAIR-1 mAb we performed an in vitro antibody dependent cell cytotoxicity (ADCC) assay with LAIR-1 expressing AML cells (MOLT4 and MV-4-11). Compared with isotype control, the LAIR-1 mAb significantly increased leukemia cell death (MV411 = 17% above isotype, and MOLT4 = 29.24% above isotype at 15 µg/ml), suggesting that the LAIR-1 mAb confers ADCC activity against LAIR1 + AML cells (Figure 2). To elucidate if the LAIR-1 mAb has a direct signaling effect on LAIR-1 + AML cells, a colony forming unit assay using primary AML cells was carried out. Interestingly, the LAIR-1 mAb inhibited colony formation by AML CD34 + cells (40-60% decreased compared with isotype control, N=4), but not normal CD34 + cells. These data suggests that our LAIR-1 mAb stimulated LAIR-1 signaling that inhibits LSC self-renewal. We then tested the in vivo anti-leukemia effect of the mAb in cell line derived xenograft (CDX) models (immune deficient mice transplanted with MV-4-11 expressing luciferase). In vivo bioluminescence imaging indicated that the LAIR-1 mAb significantly inhibited in vivo AML growth (91% reduction of total flux)(Figure 3). A significant increase in cell death was observed in the presence of the mAb in the blood (47%), spleen (89.4%) and bone marrow (27.6%). Similar to the anti-leukemic effect in CDX AML models, the LAIR-1 mAb significantly suppressed in vivo growth of AML patient derived xenografts (5 different primary AML donors) (10-90% human CD33 + AML cells in isotype control treatment vs 0.5-5% CD33 + AML cells in anti-LAIR-1 treatment, N=3) (Figure 4), while minimally impacting normal immune cells.Taken together, our studies suggest that the LAIR-1 mAb we generated is a novel AML immunomedicine that preferentially eradicates AML LSCs and blasts while preserving healthy HSCs through disruption of AML survival signals and clearance of AML through ADCP and ADCC. Additional studies are currently evaluating if this novel LAIR-1 mAb has other mechanisms of action that contribute to overall in vivo activity, including reduction of AML niche implantation, regulation of bone marrow homing and regulation of anti-tumor immunity. [Display omitted] DisclosuresTian: NextCure: Ended employment in the past 24 months. Paucarmayta: NextCure: Current Employment. Lovewell: NextCure: Current Employment. Maloveste: NextCure: Current Employment. Copeland: NextCure: Current Employment. O'Neill: NextCure: Current Employment. Patel: NextCure: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company. Myint: NextCure: Current Employment, Current holder of stock options in a privately-held company. Langermann: NextCure: Current Employment, Current holder of stock options in a privately-held company. Flies: NextCure: Current Employment, Current holder of stock options in a privately-held company. Kim: Nextcure: Research Funding.

Efficient Human Acute Myeloid Leukemia Targeting By Universal Chimeric Antigen Receptor T-Cells Via Combinatorial Use of Linkers

Chimeric antigen receptor (CAR) T-cells are genetically engineered T-cells with potent biocidal activity against respective target-expressing cells. Recently, CAR T-cells have been successfully used clinically to eradicate B cell-derived malignancies by targeting B-cell lineage specific surface antigens (e.g. CD19, BCMA). However, several limitations of current single-antigen targeting CAR T-cell therapies are becoming evident: a) low target tumor-antigen expression might lead to low CAR T-cell targeting efficacy; b) single antigen-targeting might lead to rapid selection of tumor cells with low or loss of antigen expression; c) single antigen-targeting does frequently not generate a high tumor-selectivity as tumor-antigens are frequently also expressed on healthy tissues; d) production of single-antigen CAR T-cells is time- and resource-consuming but results in effectors that target only one antigen; e) on-target off-tumor as well as off-target side-effects are difficult to control without terminally eliminating CAR T-cells in the recipient. While a-c) might be overcome by combinatorial tumor-antigen targeting, d-e) might be addressed by production of Universal CAR T-cells that recognize a specific tag on selective bridging molecules with short half-life.To address some of these limitations in principle, we have here developed universal CAR T-cells targeting fluorescein, as well as fluorescein-labeled antibody-constructs directed against several cell surface antigens, that would serve as versatile, combinatorial selective linkers and, upon target binding, also CAR T-cells activators. We then tested the system on human acute myeloid leukemia (AML) cell lines and primary patient AML blasts. Specifically, we engineered CAR T-cells, termed FluA-CAR T-cells, to display the anti-fluorescein engineered lipocalin FluA, which mediated recognition of randomly or site-specifically fluorescein-labeled antibodies in IgG or short half-life diabody (Db) format, directed against the frequently AML expressed antigens CD33, CD117 and CD371. Site-specific chemical modification methods and cysteine-tagged Db mediated the strongest AML killing results in vitro over a broad range of antibody concentrations.We then hypothesized that FluA-CAR T-cells, targeting AML cells via combinatorial use of linker molecules adapted to specific AML antigen-expression profiles, would allow to most efficiently eliminate AML cells in a dose- and timely-regulated fashion. To this end, we tested single and combinatorial use of fluoresceinated anti-CD117 Db and anti-CD371 Db on MOLM13 AML cell lines, engineered to be either CD117 highCD371 neg, CD117 negCD371 high, or CD117 highCD371 high. Indeed, combination of anti-CD117 and anti-CD371 Db-linkers, resulted in significantly improved MOLM13 cell lysis compared to equimolar concentrations of single agents in vitro (example figure). We then tested the same approach, targeting CD117 +CD371 + primary AML cells from two different patients, using either patient-derived or allogeneic FluA CAR T-cells. Again, the combinatorial use of linkers generated a significantly higher AML cell lysis than the use of single Db linkers.We thus here provide proof-of-concept for the generation of highly potent universal targeting FluA CAR T-cells from healthy donors and AML patients. By choosing suitable CAR-adaptors with respect to their conjugation chemistry and size, it is possible to tightly regulate CAR-T cell activity against CD33, CD117 and CD371 expressing AML cells and likely any tumor cell expressed antigen. Short half-life small molecule linkers will allow to control FluA CAR T-cell on-off activity and combinatorial use of linkers will allow to maximize anti-tumor activity and to minimize on-target off-tumor toxicity. [Display omitted] DisclosuresMyburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

Combining Arsenic Trioxide and Mitocans Selectively Disrupts Cellular Energetics in Acute Myeloid Leukemia

In our earlier work with arsenic trioxide (ATO) resistance in acute promyelocytic leukemia (APL), we observed that ATO resistant cells reprogrammed their metabolism from glycolysis to oxidative phosphorylation (OXPHOS) as a mechanism of resistance. We further demonstrated that it could be overcome by targeting this metabolic switch using FCCP (mitocan) in combination with ATO (Balasundaram N et al. Biorxiv 2020). There is increasing evidence that acute myeloid leukemia (AML) cells have a greater metabolic plasticity unlike ATO resistant APL cells and most cancers that rely on glycolysis. AML leukemic stem cells preferentially utilize OXPHOS for their survival (Lagadinou ED et al. Cell stem cell 2013).Mitocans like venetoclax used in combination with hypo-methylating agents are already well established in the management of AML (Pollyea D, et al. Nat Med 2021). ATO is also an effective glycolytic inhibitor (Zhang H, et al. PNAS 2015) hence we hypothesized that a combination of ATO and mitocans could potentially target the metabolic plasticity of AML cells. As the combination of ATO and FCCP was found to be non-specific we performed a small-scale screening on an AML cell line (U937) using FDA-approved compounds that are reported to target mitochondria (Gohil V et al. Nature Biotechnology, 2010). Though most of the mitocans showed predicted synergy with ATO. We focused on artesunate (ART) as a candidate for further evaluation due to its specificity for malignant cells, high therapeutic index, bioavailability, route of administration, cost-effectiveness, and global usage as an antimalarial.The combination of ATO+ART significantly reduced the viability of different subtypes of AML cell lines (THP-1, MV4:11, and Kasumi-1) and acute lymphoblastic leukemia cell lines (Jurkat E6.1, SUP B15, and MOLT-4) with minimal effect on the normal cells (CD34 and peripheral blood mononuclear cells; n=10; 48hours) (figure 1a). We noted that the selective specificity of the combination was primarily due to the iron metabolism of the leukemic cells and a requirement of iron for the activity of ART. When an iron chelator deferoxamine (DFO) was used in combination with ATO+ART there was a significant reduction in the activity of the combination on the AML cells (Figure 1b, U937; n=10; 48hours). Seahorse extracellular flux analysis validated that ART (5uM) as a single agent promoted uncoupled mitochondrial respiration and the addition of ATO resulted in a metabolic catastrophe (figure 1c and d).Chemical drug proteomic analysis using biotinylated artesunate and pull down from the leukemic cells revealed that the top interacting partners were localized in the mitochondria. We also noted that ART treatment significantly affected the mitochondrial dynamics of leukemic cells, where ART and ATO+ART treated cells had fragmented mitochondria in comparison to the control and ATO alone treated cells where the mitochondria were more elongated (figure 1e, U937 cells; n=3). We evaluated the effect of ATO+ART and their combination with azacytidine (triplet) in-vitro. Dual and triple combinations showed greater toxicity on AML cell lines and primary AML cells (Figure 1f, n=50) in comparison to the normal peripheral blood mononuclear cells (PBMNCs) and normal CD34+ cells. Taken together, these findings highlight the selective specificity of these combinations and its clinical potential in AML. [Display omitted] DisclosuresAugustin: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor. Krishna: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; KCM Vellore: Patents & Royalties; SGUL: Patents & Royalties; AML: Other: Co-Inventor. Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Tumor Associated Macrophages Promoted Fatal Patient Derived Acute Promyelocytic Leukemia In Vivo

With immune therapies on the rise, an in-depth understanding of the immunological changes in leukemic bone marrow (BM) niches becomes indispensable. Being an crucial part of the tumor microenvironment (TME) in solid tumours, tumour-associated macrophages are often associated with poor prognosis (Bruni et al. 2020). Yet, in acute myeloid leukaemia (AML) the role of macrophages has not been thoroughly studied. The expression of the M2-markers CD163 and CD206 in the AML BM cell population predicted poor clinical outcome. We identified that this expression emerges from a more mature (CD45 midSSC highHLA-DR +CD14 +CD16 +/-) myeloid cell population (hereafter called AML-associated macrophages - AAM) and not from the leukemic blasts. By employing flow cytometry analysis (FACS) we noted a decrease in the expression of the M1-marker (CD80) and an increase of the M2-markers CD163/CD206on AAM (n=70) compared to healthy donors (HD, n=10). Unsupervised clustering based on the CD163/CD206 levels detected on AAM generated 4 distinct clusters, whereby patients within the CD163 low/CD206 low cluster displayed better overall survival than the other clusters.In vitro, the co-culture of HD-derived M1 macrophages and AML primary/cell lines reduced AML growth via apoptosis induction and cell cycle arrest, while M2-macrophages promoted AML survival and phagocytosis/drug-resistance when treated with FLT3/BCL2 inhibitors. Primary AML cells were also able to repolarize M1- into M2-macrophages, suggesting that leukemic cells actively remodel their microenvironment. Next, we evaluated the impact of M2-macrophages on leukemogenesis in a patient derived xenograft (PDX) model, using the notoriously difficult to engraft primary Acute Promyelocytic Leukaemia (APL) cells (n=7 patient samples). Intra-BM injection of M2-macrophages and retro-orbital transplant of primary APL cells induced full-blown APL in NSGS mice. More strikingly, ex vivo culture of APL cells on M2-macrophages (48h) was sufficient to “train” these cells to engraft and induce fatal APL. Maintenance of self-renewal was shown in a secondary transplant and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays.To identify the biological changes acquired by leukemic blasts, we performed RNA sequencing comparing AML/APL samples at diagnosis to cells that were “trained” (48 h) on M2-macrophages or on MS5 mesenchymal BM stromal cells. Gene ontology and gene set enrichment analysis on the genes up-regulated upon M2 co-culture were significantly enriched for cell migration, cell cycle progression and oxidative phosphorylation (OXPHOS) signatures. In line with our RNAseq data, we noted improved in vivo homing of primary APL cells to the BM within 18 h post-transplant upon ex vivo M2 co-culture compared to diagnosis (n=7 APL blasts). Concurrently, we detected increased levels of surface protein expression Integrin-α4 (CD49d) and -α5 (CD49e) on APL/AML blast cells after M2 exposure. The CD49d expression remained high in primary and secondary transplants. Using seahorse measurements, we confirmed the increased respiration capacity (basal and maximum) of primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5.FACS analysis revealed that M2-macrophages were able to transfer more mitochondria than MS5 cells to primary AML cells, which could underlie the observed increase in OXPHOS mitochondrial metabolism. Treatment with Etomoxir (50 µM), prevented the gain in functional respiration when AML blast were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures, suggesting increased fatty acid oxidation to drive the OXPHO-like state. Finally, we noted that training on M2 macrophages significantly increased colony formation and endowed the cells with long term proliferation in liquid cultures for over 30 days.Overall, we reveal that the frequency of M2-macrophages is up-regulated in a subgroup of AML patients representing a group with poor prognosis. M2 macrophages can support leukemic growth and therapy-resistance, and support fatal APL in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter adhesion, homing and metabolic characteristics of leukemic blasts to allow efficient engraftment and fatal leukemogenesis. Our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions. DisclosuresSilveira: BMS/Celgene: Research Funding; Servier/Agios: Research Funding; Abbvie: Speakers Bureau; Astellas: Speakers Bureau. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding. Mota: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Technopharma: Speakers Bureau; Bristol Myer Squibb: Speakers Bureau; Bayer: Speakers Bureau; Pfizer: Speakers Bureau; AstraZeneca: Speakers Bureau; Astellas: Speakers Bureau; Ipsen: Speakers Bureau; Amgen: Speakers Bureau.

CTX-712, a Novel Clk Inhibitor Targeting Myeloid Neoplasms with SRSF2 Mutation

Splicing factors (SFs) are among the most frequent mutational targets in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and a subset of acute myeloid leukemia (AML), designated as chromatin/spliceosome-mutated AML, where major SFs mutated include SF3B1, SRSF2, U2AF1, and ZRSR2. SF mutations are largely mutually exclusive and except for ZRSR2 mutations, heterozygous, suggesting synthetic lethality of homozygous or multiple mutations. Thus, SF functions might be a plausible target of therapy for MDS/AML. Of potential interest in this regard is serine/arginine-rich (SR) domains ubiquitously shared by many SFs, including U2AF1, SRSF2, and ZRSR2, which need to be phosphorylated for their nuclear translocation by evolutionally conserved kinases, known as CLK family of proteins. CLK family kinases regulate mRNA splicing by phosphorylating various SR proteins, and inhibition of CLK family kinases resulted in reduction of phosphorylation levels of SR proteins, induction of splicing alterations and protein depletion for multiple genes. In addition, a recent report showed that CLK inhibition can induce skipped exons, cell death, and cell growth suppression. Thus, CLK inhibitors might have a role in the therapeutics of SF-mutated MDS/AML, by further compromising RNA splicing.We have recently developed an orally available and highly potent CLK inhibitor, CTX-712, and evaluated its anti-leukemic activities both in vitro and in vivo. When tested in human myeloid cell lines (K562 and MV-4-11), CTX-712 showed a strong inhibitory effect on cell proliferation (IC 50=0.15 and 0.036 μM, respectively). The anti-leukemic effect was also confirmed by survival assay using a total of 79 primary AML cells (the average of IC 50 was 0.078 μM). In addition, CTX-712 suppressed phosphorylation of multiple SR proteins including SRSF3/4/6, all of which bind to SRSF2. RNA-seq analysis revealed that CTX-712 induced global splicing changes, which typically resulted in skipped exon. Notably, the degree of splicing (percent spliced-in value) in skipped exon events induced by the drug was positively correlated with the sensitivity to the drug (IC 50) in primary AML cells (n=32, R=0.61, P=0.00018).To further investigate the effect of CTX-712 on tumor growth in vivo, we established 13 MDS/AML-derived xenografts (PDXs), which were treated with varying doses of CTX-712. Among the 13 PDX models, SRSF2 mutation was found in 2 cases, which had the SRSF2 P95H or P95L mutation. The SRSF2 P95H PDX showed a significant response to CTX-712 in a dose-dependent manner. Of note, 4 out of 5 mice treated using a high dose protocol (12.5 mg/kg) achieved complete remission (the tumor shrank completely to unmeasurable size). Two weeks after treatment, tumor volumes (mm 3) were 762 ± 147 (vehicle), 331 ± 64 (low dose of CTX-712: 6.25mg/kg, P=0.0395), and 39 ± 39 (high dose, P=0.0064) (N=5 each, mean ± SEM). Interestingly CLK inhibition induced aberrant splicing events including skipped exons in vivo, which were more strongly affected in the SRSF2-mutated model. In addition, CTX-712 efficacy was also confirmed in the model with the SRSF2 P95L mutation. The SRSF2 P95L model showed a significant reduction in tumor volumes (mm 3) 2 weeks after CTX-712 treatment; 406 ± 94 (vehicle) and 75 ± 17 (high dose, P=0.0162) (N=6 each, mean ± SEM). CTX-712 also significantly improved the survival of the SRSF2 P95L-mutated model (high dose, P=0.0030) (N=6 each).Overall, 10 out of 13 PDX AML/MDS models, including 2 SRSF2-mutated models, showed anti-tumor effect of CTX-712. Complete disappearances of tumors were obtained in the SRSF2 P95H mutation model. These results provide mechanistic insights of CLK inhibition and a rationale for further investigation of the novel CLK inhibitor in MDS/AML. DisclosuresYoda: Chordia Therapeutics Inc.: Research Funding. Morishita: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Mizutani: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Tozaki: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Satoh: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Miyake: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Ogawa: Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company.

Associating Ex Vivo Drug Sensitivity with Differentiation Status Identifies Effective Drug Combinations for Acute Myeloid Leukemia

The development of molecularly-targeted therapies to improve outcomes relative to chemotherapy for acute myeloid leukemia (AML) is impeded by the heterogeneity of genetic aberrations that contribute to disease. Among the multitude of biological mechanisms that lead to AML disease initiation and progression is dysregulation of cytokine signaling pathways, a hallmark of chronic inflammation, which contribute to the growth, survival, and differentiation state of AML cells. We have previously shown that IL-1β, a pro-inflammatory cytokine expressed by many cell types including macrophages and monocytes, stimulates proliferation of leukemic blasts independent of mutational status in primary AML samples via enhanced phosphorylation of p38α MAPK, an effect that can be blocked by IL-1 receptor knockdown or by pharmacologic inhibition (Carey 2017). Additionally, recent studies have shown sensitivity to the approved BCL2 inhibitor venetoclax in AML associates with undifferentiated leukemic cells (Pei 2020; Zhang 2018; Majumder 2020). Based on these associations, we evaluated the combination of doramapimod (DORA), a p38 MAPK inhibitor, with venetoclax (VEN) for potential enhanced sensitivity on primary AML cells. Ex vivo drug screening of primary AML patient samples (n=335) revealed significantly enhanced efficacy of VEN+DORA compared to either single agent (Nemenyi test; p<0.0001). This broad sensitivity of the VEN+DORA combination was not significantly associated with an array of clinical, genetic, and mutational features in the patient samples tested, in contrast to single agents, particularly VEN. Analysis of blood cell differential counts of patient samples tested identified increased monocyte levels were significantly correlated with sensitivity to DORA and resistance to VEN as single agents (Spearman r = -0.3 and 0.6; p<0.0001), associations that were not apparent with the combination. For patient samples with accompanying FAB differentiation state-based designations (n=108), sensitivities of the combination were similar across classifications of undifferentiated (M0/M1) through monocytic (M4/M5) acute leukemia. In contrast, single-agent VEN was significantly more sensitive in undifferentiated compared to monocytic specimens, whereas DORA sensitivity showed the reverse trend (though to a lesser degree). These differences in sensitivity were further validated by immunophenotyping data where available (n=105), which showed surface markers associated with resistance to VEN (CD11b, CD14, CD16, CD56, CD64, HLADR; Wilcoxon Rank Sum, p<0.001 to p=0.007) or sensitivity to VEN (CD117; p=0.001) or DORA (CD14; HLADR; p=0.004). By contrast, none of these associations significantly distinguished sensitivity for the VEN+DORA combination. Expression levels of MAPK14 and BCL2, the respective primary targets of DORA and VEN, were concordant with their respective drug sensitivities associated with FAB classification; that is, significantly higher levels of BCL2 in M0/M1 leukemias and MAPK14 in M4/M5 cases (Mann-Whitney test; p<0.0001; n=145). Further dissection of transcriptomic and drug sensitivity data revealed strong correlation and gene set enrichment for DORA and VEN sensitivities with monocyte-like and progenitor-like signatures, respectively (n=225), for cell differentiation states previously described for AML (van Galen 2019), and these associations diminished for the combination treatment. Lastly, the VEN+DORA combination enhanced efficacy and synergistic inhibition was confirmed using human AML cell line models tested with a matrix of potential dose concentrations. Taken together, these findings suggest that exploiting distinct, complementary sensitivity profiles of targeted therapies with respect to leukemic differentiation state, such as dual targeting of p38 MAPK and BCL2, offers an opportunity for broad, enhanced efficacy across the clinically challenging heterogeneous landscape of AML. DisclosuresDruker: Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Recludix Pharma, Inc.: Consultancy; EnLiven: Consultancy, Research Funding; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Aileron: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Nemucore Medical Innovations, Inc.: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Genentech: Research Funding; Takeda: Research Funding; Astrazeneca: Research Funding; Constellation: Research Funding; Agios: Research Funding; Petra: Research Funding; Incyte: Research Funding; Array: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Seattle Genetics: Research Funding; Schrodinger: Research Funding.

Inhibiting Mitochondrial Complex II Exposes a Novel Metabolic Vulnerability in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematological malignancy, characterized by an increased reliance on mitochondria-related energetic pathways including oxidative phosphorylation (OXPHOS). Consistent with this, the electron transport chain (ETC), a component of OXPHOS has been demonstrated to be a suitable anti-leukemia target, with ETC complex I inhibitors currently in clinical development. Relative to its counterparts, complex II (CII) is unique in that it directly links the ETC to the tricarboxylic acid (TCA) cycle through succinate dehydrogenase (SDH) activity. Moreover, it is the only ETC complex with elevated activity in AML, relative to normal hematopoietic samples, with indirect inhibition selectively targeting AML cells. However, direct CII inhibition in AML has not been previously investigated, nor have the mechanisms underlying the divergent fates of AML and normal cells upon CII inhibition.A genetic approach was first used to assess the effects of CII impairment on AML growth in vitro and in vivo. Using lentiviral mediated shRNA we generated AML cell lines lacking succinate dehydrogenase assembly factor 1 (Sdhaf1). Sdhaf1 knockdown suppressed CII activity, cell proliferation and clonogenic growth across all three cell lines and delayed leukemia growth in vivo. To recapitulate these effects through a pharmacological approach, we aimed to identify a novel CII inhibitor, since currently available inhibitors are only effective at high doses and are neurotoxic. Through an in silico structural screen and molecular docking study, shikonin was identified as a small molecule that selectively binds to CII. Shikonin inhibited CII activity in the AML cells lines and patient-derived samples, and selectively killed AML cells (EC 50: 1.0μM ± 0.04) while sparing normal progenitors. In murine engraftment models, shikonin (2.0-3.0 mg/kg, 3x/week for 5 weeks) significantly reduced engraftment of patient-derived AML cells but had no effect on normal hematopoiesis.To further characterize the mechanisms governing the divergent cell fates of CII inhibition, we performed stable isotope metabolic tracing using 13C 6- glucose and 13C 5, 15N 2-glutamine in patient-derived AML cells and normal mobilized peripheral blood mononuclear cells (MNCs). Both pharmacological and genetic loss of CII resulted in TCA cycle truncation by impairing oxidative metabolism of both glucose and glutamine. In Sdhaf1 knockdown and primary AML cells, this led to a depletion in steady state levels of TCA metabolites proceeding SDH. Inhibition of CII most notably suppressed levels of aspartate, a nucleotide precursor whose levels dictate the proliferative capacity of a cell under ETC dysfunction. Remarkably, MNCs maintained aspartate levels despite inhibition of CII, which was attributed to reductive carboxylation of glutamine, an alternate metabolic pathway that can regenerate TCA intermediates when OXPHOS is impaired. In contrast, while reductive carboxylation was also active in AML cells after CII inhibition, this activity was insufficient to maintain aspartate levels and resulted in metabolite depletion and cell death. Thus, loss of CII activity results in diverse cell fates whereby normal haematopoietic, but not AML cells sufficiently use reductive carboxylation of glutamine to overcome TCA cycle truncation, sustain aspartate levels and avert cell death. This is further evident through modulation of glutamine entry into the TCA cycle, where supplementation of cell-permeable α-ketoglutarate abrogated shikonin-mediated cell death while concomitant treatment with the glutaminase inhibitor CB-839, sensitized cells.Together, these results expose reductive carboxylation to support aspartate biosynthesis, as a novel metabolic vulnerability in AML that can be pharmacologically targeted through CII inhibition for clinical benefit. DisclosuresMinden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Vitamin C Enhances PARPi Efficacy for the Treatment of AML

Poly-ADP-ribose polymerase inhibitors (PARPi) are currently in clinical trial to determine their therapeutic efficacy for the treatment of acute myeloid leukemia (AML). We have shown that vitamin C (VitC), an essential micronutrient and co-factor of Ten-Eleven translocation (TET) proteins, enhances AML sensitivity to PARPi, potentially due to an increased dependency on base-excision repair (BER) enzymes needed to remove TET-catalyzed oxidized methylcytosine bases via active DNA demethylation. TET2 is the most frequently mutated TET gene in patients with AML, and vitamin C treatment can mimic genetic restoration of TET2 function, leading to DNA demethylation, differentiation, and leukemia cell death. Whether vitamin C efficacy in combination with PARPi depends on the level of TET2 functional alleles is not yet known and may stratify whether TET2 wild-type or mutant patients should be targeted by vitamin C adjuvant therapy.We have generated primary murine AML-ETO9a+ and MLL-AF9+ leukemia models with Tet2 +/+, Tet2 +/- and Tet2 -/- alleles to determine the Tet2-dependent efficacy of PARPi treatment when combined with vitamin C. Furthermore, we have performed CRISPR gene knockout and drug library screening in human AML cell lines in combination with vitamin C treatment, and tested a panel of 10 AML cell lines with titrating concentrations of PARPi (Olaparib, Talazoparib, Veliparib and Rucaparib) alone or in combination with vitamin C (L-ascorbic acid) mimicking physiological to pharmacological in vivo doses. Primary murine AML cells and human cell lines were assayed for colony-forming capacity, differentiation, cell cycling, viability and effects on DNA methylation, levels of oxidized 5-mC and gene expression upon combination treatment in vitro and in vivo. TET2 mutant PDX and primary murine AMLs treated in vivo with L-ascorbate (4g/kg) and Olaparib (50mg/kg) by daily IP injection were also monitored for disease burden, cellular differentiation and survival.Vitamin C is known to drive the TET-catalyzed iterative oxidation of 5-methylcytosine (5-mC) leading to the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). We show that VitC-PARPi combination treatment causes an accumulation of oxidized 5-mC intermediates in the AML genome that correlates with increased yH2AX formation in mid-S phase and cell cycle stalling. Vitamin C reduces the IC 50 of Olaparib and Talazoparib by greater than 10-fold in human AML cells lines and primary murine leukemia cells, and treatment in combination promotes myeloid differentiation and blocks colony-forming capacity greater than either alone. In both our in vitro and in vivo studies, Tet2 +/- AML cells exhibit increased sensitivity to vitamin C treatment alone or in combination with PARPi compared to either Tet2 +/+ or Tet2 -/- cells, suggesting that patients with TET2 haploinsufficiency, which represents the majority of TET2 mutant cases, could benefit the most from combined treatment.Our findings confirm that vitamin C can act synergistically with PARPi to block AML cell viability, reduce colony-forming capacity, and decrease leukemia burden in PDX and primary murine leukemia models in a TET2 allelic dose-dependent manner. The combinatorial effect works at clinically relevant concentrations of PARPi, and low-pharmacological doses of vitamin C. These studies suggest that vitamin C can be used as a non-toxic therapeutic adjuvant to PARPi therapy for the treatment of AML. DisclosuresNeel: Northern Biologics, LTD: Current equity holder in publicly-traded company, Other: Co- Founder; SAB: Other: Co-Founder; Navire Pharma: Consultancy, Current equity holder in publicly-traded company; Jengu Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Co-Founder; Arvinas, Inc: Consultancy, Current equity holder in publicly-traded company; Recursion Pharma: Current equity holder in publicly-traded company.

Acute Myeloid Leukemia Cell-Intrinsic PD-1 Functions Promoting Leukemia Development By Recruiting SHP2

Acute myeloid leukemia (AML) is a devastating hematopoietic malignancy. With current therapies, only approximately 30% of patients achieve long-term survival. Therefore, novel, more active and less toxic treatments are urgently needed. Programmed death-1 (PD-1) is a cell surface receptor that functions as a T cell checkpoint and plays a central role in regulating T cell exhaustion. Binding of PD-1 to its ligand, programmed death-ligand 1 (PD-L1), activates downstream signaling pathways and inhibits T cell activation. Abnormally high PD-L1 expression on tumor cells and antigen-presenting cells in the tumor microenvironment mediates tumor immune escape, and PD-1/PD-L1 immune checkpoint blockade has showed promising results in cancer patients. Recently, PD-1 expressed on melanoma cells was also shown to play a pivotal role in tumor growth. To date, in AML, the function of PD-1 has been mainly studied in the host T cells, while little is known regarding the role of PD-1 in AML cells. Herein, we examined the level and role of PD-1 in AML cells using AML murine model and patient samples.We used MLL PTD/WT/Flt3 ITD/ITD knock-in mouse in B6 background, a well characterized AML model, to study the expression and function of PD-1 in AML. Flow cytometric analysis of LSK (Lin -Sca-l1 +c-kit +) cells from the bone marrow (BM) of wild-type (WT, n=5) and AML (n=10) mice showed that 20.9%-61.9% of AML LSKs versus (vs) <5.0% of normal LSKs are PD-1 positive (P< 0.0001). Western blot and Q-RT-PCR analysis confirmed higher levels of PD-1 in AML LSKs than in normal LSKs. The PD-1 levels on LSKs increased over time and associated with disease progression. Similar results were obtained in AML patients, showing PD-1 + in 0.2%-14.6% of AML CD34 + cells vs < 1.0% of normal CD34 + cells (P< 0.05). Data analysis based on TCGA showed that higher PD-1 levels are associated with shorter survival in AML patients (P=0.0125).To assess the functional role of PD-1 in AML, we sorted PD-1 + and PD-1 - fractions fromAML LSKs and observed a lower frequency of quiescent cells (G0, 16.60% vs 44.87%, P< 0.05) and a higher cell growth rate in the PD-1 + vs PD-1 - cells. Further in vivo study showed that PD-1 + AML LSKs (CD45.2) generated higher white blood cell (WBC) counts (P< 0.0001), higher AML engraftment (P< 0.0001) and a shorter survival (median survival 57.5 vs >75 days, P< 0.001) in recipient mice (CD45.1) compared with PD-1 - AML LSKs. Similar results were observed in human samples. Compared to PD-1 - CD34 + cells, PD-1 + CD34 + cells are less quiescent and more proliferative (P< 0.01). PD-1 + AML blasts had higher engraftment rate (13.18% vs 2.68%, p=0.0002) and shorter survival (median survival: 27 vs 45 days, P= 0.0008) in NSGS mice than PD-1 - AML blasts.To evaluate if these in vivo differences observed in PD-1 + vs PD-1 - AML LSKs were mediated by interactions between PD-1 + AML and T cells, PD-1 + and PD-1 -LSKs from AML mouse were transplanted into T-, B- cell-deficient NSG mice. Recipient mice transplanted with PD-1 + AML LSKs had higher WBC counts (P< 0.01), higher AML engraftment (P< 0.0001) and a shorter survival (median survival: 76 vs >130 days, P< 0.0001) than recipients with PD-1 - AML LSKs, suggesting that these differences were T cell-independent.Next, we examined whether blocking PD-1 could affect leukemic cell growth. We sorted LSK cells from AML mice and performed colony forming cell (CFC) assay in the presence of anti-PD1 antibody or isotype antibody. Blocking PD-1 with anti-PD-1 antibody significantly suppressed CFC and cell growth in vitro but did not induce apoptosis compared to isotype control antibody. To explore the molecular mechanism by which PD-1 contributes to AML growth, we then sorted PD-1 + and PD-1 - LSKs from AML mice for molecular studies. Western blot assays revealed higher levels of SHP-2 and phosphorylated (p) -ERK in PD-1 + vs PD-1 - AML LSKs. We validated these results in primary human AML cells by immunofluorescence staining. Confocal microscopy of PD-1 + and PD-1 - human AML CD34 +cells demonstrated that PD-1 localized at the cell membrane and in the cytoplasm and p-ERK was markedly enhanced in the PD-1 + CD34 + cells.In conclusion, we showed here that a subpopulation of murine and human AML blasts expresses high levels of PD-1 which mediated disease initiation and growth through activation of the MAPK/ERK signaling pathways. PD-1 blocking antibody reversed these activities and might contribute to the clinical efficacy of anti-PD-1 therapy in AML. DisclosuresMarcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

CD33 BiTE ® Construct Mediated Immunological Synapse Formation and Downstream Signaling in T Cells Is Dependent on Expression of Costimulatory Molecules on Target Cells

CD33 BiTE ® Construct Mediated Immunological Synapse Formation and Downstream Signaling in T Cells Is Dependent on Expression of Costimulatory Molecules on Target Cells

Deficiency of the E3 Ligase Hoip Impairs Adult Hematopoiesis and Myeloid Leukemia

Ubiquitination is a post-translational modification that plays important roles in the regulation of various cellular processes. The linear ubiquitin chain assembly complex (LUBAC) is the only E3 ubiquitin ligase, which can specifically generate linear ubiquitin chains. LUBAC is composed of three subunits: catalytic Hoip (Rnf31), Hoil-1l (RBCK1), and Sharpin. LUBAC regulates canonical NF-κB signaling pathway and apoptosis. Several studies have shown that LUBAC is required for fetal hematopoiesis and tumorigenesis in several solid cancers, but the role of Hoip, which is the catalytic component of the LUBAC, in adult hematopoiesis and myeloid leukemia is unclear.To address whether Hoip is required for adult hematopoiesis, we used tamoxifen induced Hoip deleted mice (Hoip fl/fl; Rosa26-CreERT). Deletion of Hoip reduced the numbers of almost all cell fractions in bone marrow and peripheral blood, except for long-term hematopoietic stem cell (LT-HSC) fraction (CD34 - Flk2 - Lineage - c-Kit + Sca-1 +: LSK). Deletion of Hoip treated with tamoxifen markedly impaired colony formation of LT-HSCs in vitro. Among competitive transplantation assay using bone marrow cells from Hoip fl/fl; Rosa26-CreERT mice, deletion of Hoip treated with tamoxifen following stable reconstitution 8 weeks after transplantation led to a significant reduction of Hoip-deficient chimerism compared to control in vivo (Figure A). Deletion of Hoip induced apoptosis in bone marrow cells and increased the frequency of CD34 - LSK cells in S/G2/M phase of the cell cycle. Collectively, these data indicated that Hoip is required for adult hematopoiesis.To evaluate the consequences of Hoip deletion in myeloid leukemia propagation and maintenance, we utilized aggressive murine myeloid leukemia models driven by retroviral transduction of oncogenes: MLL-AF9 and NRAS G12V-driven acute myeloid leukemia (AML) and BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) models. Deletion of Hoip in established leukemia cells treated with tamoxifen markedly impaired colony formation of leukemia cells in vitro and led to a significantly longer survival with reduced disease burden in bone marrow and spleen in vivo in both of leukemic models (Figure B,C). Deletion of Hoip also induced apoptosis in leukemia stem cells (LSCs: c-Kit + Lineage - cells) (Figure D) and increased the frequency of LSCs in S-phase of the cell cycle. Furthermore, we evaluated the contribution of HOIP to human myeloid leukemia. Knockdown of HOIP by small hairpin RNA using lentivirus in several myeloid leukemia cell lines (THP-1, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro, and increased apoptosis. LUBAC inhibitor gliotoxin impaired the growth of murine and human leukemia cells. These results indicate that Hoip is essential for propagation and maintenance in murine and human myeloid leukemia.Because LUBAC regulates canonical NF-κB signaling pathway, we evaluated protein levels of p65 phosphorylation on Ser536 (p-p65) and IκBα in leukemia cells by flow cytometric analysis. Reduction in p-p65 and inhibition of IκBα degradation were observed in Hoip-deficient leukemia cells (Figure E), indicating that Hoip plays an important role in regulation of NF-κB signaling in myeloid leukemia.Since Hoip is the central component of LUBAC and the only catalytically active subunit of E3 ubiquitin ligase, we examined the importance of each domain of Hoip for myeloid leukemia propagation using Hoip mutant isoforms. Loss of colony-forming ability of Hoip-deficient AML cells could not be rescued by retroviral transduction of the Hoip mutants with deletion of UBA domain (which is required for interaction with the other LUBAC subunits) and with deletion of RBR domain and with mutation in C879A (which lack ligase activity) (Figure F), indicating that LUBAC ligase activity and interaction with the other LUBAC subunits are critical for leukemia propagation.Thus, our data demonstrated that Hoip is essential for adult hematopoiesis and myeloid leukemia. Given that patients with myeloid leukemia have shown increased activity of NF-κB signaling, inhibition of LUBAC ligase activity could serve as a promising strategy for treating myeloid leukemia. [Display omitted] DisclosuresJimbo: Japan Society for the Promotion of Science: Research Funding. Ito: Japan Society for the Promotion of Science: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Iwama: Nissan Chemical Corporation: Research Funding. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Konuma: Japan Society for the Promotion of Science: Research Funding; The Japanese Society of Hematology: Research Funding; SGH Foundation: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

A Bispecific Antibody Targeting CD117 and CD3 Enables T Cell Mediated Killing of CD117-Expressing Healthy and Malignant Hematopoietic Cells

INTRODUCTIONHematopoietic stem and progenitor cells (HSPCs) support life-long hematopoiesis. A single HSPC can also be at the origin of hematological malignancies, such as Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS). Allogeneic HSCT with the intent to eliminate recipient AML or MDS and at the same time replace recipient HSPC with donor-HSPC and immune cells is a life-saving therapeutic option for many patients. However, chemotherapy (and sometimes in addition gamma-irradiation based conditioning regiments) prior to HSCT are associated with substantial toxicity. Thus, due to benefit-outweighing treatment-related toxicity and mortality, frail, multi-morbid and elderly patients are usually excluded from potentially curative allo-HSCT approaches. For these reasons, more selective preconditioning strategies, leading to residual AML/MDS elimination and creating “space” for incoming HSPCs, are required. Selective targeting of CD117 with monoclonal antibodies has been proposed as a strategy to remove endogenous HSPCs, enabling an effective but mild preconditioning. However, specific conditioning of AML and MDS patients, prior to HSCT, might require a more potent effector cell type. We hypothesized that a CD117 and CD3 binding, T cell engaging and activating antibody construct (CD117xCD3 TEA) with a short half-life might be an ideal means to selectively eliminate CD117-expressing healthy HSPCs and residual CD117-expressing AML or MDS cells prior to allo-HSCT. METHODSWe cloned and expressed CD117xCD3 TEA in tandem scFv format and produced it by transient gene expression in Chinese hamster ovary cells (CHO-S). The fusion proteins were purified to homogeneity by protein A affinity chromatography. We derived target cell lines with varying surface levels of CD117 (high, medium and low) from CD117 negative parental cell lines HL-60 and MOLM-14 (Myburgh et al., Leukemia, 2020). To assess T cell mediated killing of target cells, we mixed them with human T cells (purified and enriched after negative selection) at varying Effector-to-Target (E:T) cell ratios and added CD117xCD3 TEA at different concentrations. The mixture was incubated and specific killing was quantified via flow cytometry at different time-points. RESULTSIn order to characterize the biocidal properties of CD117xCD3 TEA, we performed in vitro killing experiments against cell lines, HSCPs from healthy donors and blast cells from AML patients. A dose-dependent in vitro killing of the cell lines was observed in the presence of various concentrations of CD117xCD3 TEA and of human T cells at an E:T cell ratio of 10:1 after 24h. The HL60 CD117 high cell line was efficiently lysed (~90%) at 100 ng/ml of CD117xCD3 TEA, corresponding to ~1.8 nM. In similar experiments with different E:T cell ratios, we observed that both HL60 CD117 high and CD117 medium cells could be quantitatively killed at E:T ratios as low as 1:1, while the killing of CD117 low cells required a higher density of T cells. The biocidal effect on non-transduced HL60 cells was negligibly low, confirming the requirement of a simultaneous engagement of CD117 and CD3 for specific killing. We repeated the same experiment with an engineered MOLM14 cell line, which also expressed CD117 at comparable high levels, incubating the target cell line with human T cells at an E:T of 1:1 for 24, 48 or 72, 120 or 192 hours. Complete killing of the target cell line was achieved at 120 and 192 hours and after supplemental addition of T cells and CD117xCD3 TEA at 72 hours (see example figure). Experiments with primary cells (HSPCs from healthy donors or blast cells from AML patients) at an E:T of 1:1 confirmed specific killing of target cells in an antigen-density- and concentration-dependent manner after 48h. CONCLUSIONSWe have generated a novel bispecific antibody, which binds to human CD117 (expressed on HSCPs and AML/MDS blast cells) and to CD3 (expressed on T cells), which we term CD117xCD3 TEA. The antibody induces selective T cell-mediated killing of cell lines with different surface levels of CD117, as well as of healthy HSPCs and primary human AML cells. Thus, the newly generated CD117xCD3 TEA might be developed clinically in order to erradicate residual AML/MDS and at the same time serve as a milder preconditioning approach prior to allo-HSCT in frail AML/MDS patients. [Display omitted] DisclosuresKiefer: ETH Zurich: Current Employment, Patents & Royalties: CD117xCD3 TEA. Myburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Guggisberg: F. Hoffmann-La Roche AG: Current Employment. Abdelmotaleb: F. Hoffmann-La Roche AG: Current Employment. Mock: Philogen S.p.A.: Current Employment. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: University of Zurich: Patents & Royalties: CD117xCD3 TEA; CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company.

Newly Developed Oncolytic Herpes Simplex Viruses Expressing Multiple Immunomodulatory Transgenes Effectively Target AML Cells

Acute myeloid leukemia (AML) is the most common human leukemia and is a major area of unmet medical need among hematologic malignancies. Progress has been made in identifying therapeutic targets and several approved therapies, but resistance to frontline chemotherapy remains a major cause of treatment failure, highlighting the need for new therapies. Oncolytic viruses (OV) are a promising new class of therapeutics that rely on tumor specific oncolysis and the generation of a potent adaptive anti-tumor immune response for efficacy. To investigate if our newly developed oncolytic herpes simplex viruses (oHSVs), designed to potentiate anti-leukemia immunity, effectively target primitive AML cells, we evaluated oHSV-VG161, which is engineered to express IL-12, IL-15 and the IL-15 receptor alpha subunit, along with a peptide fusion protein capable of disrupting PD-1/PD-L1 interaction. After screening several AML cell lines that expressed relatively high levels of a HSV entry receptor (HVEM), we demonstrated that VG161-infected OCIAML3 and MOLM13 cells significantly enhanced cell killing (IC 50: 0.4 & 1.8 multiplicity of infection (MOI) as compared to MV4-11 and U973 cells (IC 50: 3.0 & 9.5 MOI). These effects were 2-3 folds lower in control VG160-infected cells. We also observed that VG161-infected AML cells induced apoptosis in a dose-dependent manner (~50%) after 48 hours and cleaved PARP, Caspase-3 and Caspase-8 were increased in these cells, and to a lesser extent in control VG160-infected cells. Both VG160 and VG161 viruses replicated efficiently in OCIAML3 and MOLM13 cells in a timely, dose-dependent manner, evidenced by qPCR detection of HSV-1 ICP27 DNA copy numbers (>500-fold increase) over 48 hours of treatment. This result was supported by detection of protein expression of HSV-1 glycoprotein D in VG160 and VG161-infected cells (up to 40% of protein detected) by FACS analysis. Interestingly, IL-12 but not IL-15 protein expression was found in intracellular-stained VG161-infected OCIAML3 and MOLM13 cells in a dose-dependent manner (up to 13% of protein detected, P<0.01) but not in VG160-infected cells, as assessed by FACS analysis. Production of IL-12 was also detected in cultured media obtained from VG161-infected AML cells (up to 150 pg/mL) by ELISA. To investigate potential molecular mechanisms of VG161-mediated anti-leukemia response and specific signalling pathways, we have screened several potential candidates and found immune regulating genes, such as IRF3, IRF7, IRF9, NFkB and ISGs, as well as type I IFN to be highly increased in VG161-infected cells as compared to VG160-infected cells (2-4-folds, P<0.001) in a dose dependent manner over 48 hours of treatment, assayed by qRT-PCR. Western blot analysis demonstrated increased phosphorylation of p-STAT1 and its protein expression in VG161-infected cells compared to VG160 control cells (~2-fold). These results suggest that VG161 viruses expressing several engineered immunomodulatory transgenes, particularly IL-12, contribute to anti-leukemia responses by activating specific immune regulating pathways such as the JAK/STAT pathway. In addition, we detected an increase in both RNA and protein levels of PD-L1 in VG161-infected AML cells, suggesting the necessity of PD-L1 blocking peptide in the viral construct. To further investigate VG161's role in regulating innate and adaptive immune responses, we have examined the biological effects of VG160/VG161 in the presence of healthy peripheral blood mononuclear cells (PBMC) in both AML cell lines and primary AML patient cells in vitro. Most interestingly, VG160 or VG161-infected OCIAML3 and MOLM13 cells show enhanced cell killing when co-cultured with PBMC and this cell killing effect was greatly enhanced in VG161-infected cells as compared to VG160-infected cells, especially in the MOLM13 cell line (up to 90% killing). This observation was further supported when primitive AML patient cells were co-cultured with VG161 and PBMC as compared to VG160 control cells. Moreover, PD-L1 expression was highly increased in AML patient cells when cultured with VG161 as compared to VG160 (2.7-fold) and this was further enhanced when co-cultured with VG161 and PBMC. Thus, we have demonstrated that newly developed oHSVs engineered with several immunomodulatory transgenes effectively target primitive AML cells, suggesting a potential treatment strategy for AML. DisclosuresNo relevant conflicts of interest to declare.

Augmenting Efficacy of T-Cell Bispecific Antibodies in AML through a Tumor Stroma-Targeted 4-1BB Agonist

Bispecific antibodies represent a promising treatment option for acute myeloid leukemia (AML). We have recently described a novel T-cell bispecific antibody (TCB) targeting the intracellular tumor antigen Wilms tumor 1 (WT1) in the context of HLA-A*02 (Augsberger et al. Blood 2021). Based on these findings a multicenter first-in-human clinical trial was initiated in relapse/refractory AML (NCT04580121). Possible immune escape mechanisms against T-cell based immunotherapy are provided by the tumor microenvironment (TME) of the bone marrow by co-inhibition of T cells or stromal cells shielding leukemic cells from immune effector cells. To overcome the immunosuppressive effect of the TME and to enhance T-cell responses, we evaluated the combination of the WT1-TCB with an antibody fusion protein that targets a stromal antigen (Fibroblast-activation protein; FAP) and provides a positive costimulatory signal (4-1BBL) to T cells. FAP is upregulated on cancer-associated fibroblasts after remodulation of the bone marrow niche by leukemic cells, and the FAP specificity of the molecule therefore provides T-cell co-stimulation tightly restricted to the tumor niche.Efficacy of the combination (WT1-TCB + FAP-4-1BBL antibody fusion protein) was evaluated in co-culture assays over 4 days with primary HLA-A*02 + AML cells, healthy donor (HD) T cells and three NIH-3T3 fibroblast cell lines. NIH-3T3 cell lines were genetically modified to express low and high levels of FAP, respectively. Wild-type NIH-3T3 cells were included as control. Additionally, a control (Ctrl)-TCB and a Ctrl-4-1BBL antibody fusion protein recognizing a non-tumor target derived from the human germline repertoire were included. Enhancement of T-cell mediated cytotoxicity by the FAP-4-1BBL antibody fusion protein was evaluated by (1) specific lysis of primary AML cells, (2) upregulation of the T-cell activation markers CD25 and 4-1BB, (3) T-cell expansion calculated as fold change compared to day 0, and (4) Granzyme B-expression which was evaluated by intracellular staining.After 4 days of co-culture, with an E:T ratio of 1:2, we observed a mean specific lysis of 55.1±8.2% (±SEM; n=4) of primary AML cells mediated by HD T cells and WT1-TCB. Notably, this was reduced to 19.4±5.9% (±SEM; n=4) in the presence of NIH-3T3 cells. However, AML cell lysis was restored by the addition of the FAP-4-1BBL antibody fusion protein in the presence of high FAP expressing NIH-3T3 cells (mean specific lysis: 62.8±7.3%; ±SEM; n=4). Concomitantly, the FAP-4-1BBL antibody fusion protein led to increased expression of the activation molecules CD25 (MFI ratio: 22.1±5.3 vs. 10.4±1.3; ±SEM; n=4) and 4-1BB (MFI ratio: 10.4±6.0 vs. 2.1±0.3; ±SEM; n=4) on CD3 + T cells. Furthermore, lysis was accompanied by increased frequencies of granzyme B expressing T cells (45.0±2.5% vs. 16.1±5.3%; n=3). Importantly, the FAP-4-1BBL antibody fusion protein led to improved T-cell proliferation, especially of CD8 + T cells (fold change on day 4 vs day 0: 5.7±2.2 vs. 1.0±0.3; ±SEM; n=4). Overall similar observations were made in the presence of low FAP expressing NIH-3T3 cells.Taken together, we have established an in vitro model system mimicking the immunoprotective bone marrow TME using NIH-3T3 cells resulting in impaired AML cell lysis. Providing additional T-cell co-stimulation by a tumor-stroma targeted 4-1BB agonist, however, restored WT1-TCB-mediated cytotoxicity of primary AML cells in the presence of FAP expressing cell lines. Importantly, the combination overcame the immunosuppressive effect of the NIH-3T3 cells on T cells as further demonstrated by improved T-cell activation and expansion. The tumor-stroma targeted 4-1BB agonist therefore represents a promising combinatorial approach to enhance T-cell activity at the local tumor site and warrants further investigations in an in vivo model system. DisclosuresPulko: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Claus: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Buecklein: Pfizer: Consultancy, Honoraria, Speakers Bureau; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Miltenyi: Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Umana: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Klein: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; Klinikum der Universität München: Current Employment; Roche: Research Funding; Seattle Genetics: Consultancy, Research Funding; Pfizer: Consultancy, Speakers Bureau; Janssen: Consultancy; Takeda: Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

Mitochondrial inhibitors circumvent adaptive resistance to venetoclax and cytarabine combination therapy in acute myeloid leukemia.

Therapy resistance represents a major clinical challenge in acute myeloid leukemia (AML). Here we define a 'MitoScore' signature, which identifies high mitochondrial oxidative phosphorylation in vivo and in patients with AML. Primary AML cells with cytarabine (AraC) resistance and a high MitoScore relied on mitochondrial Bcl2 and were highly sensitive to venetoclax (VEN) + AraC (but not to VEN + azacytidine). Single-cell transcriptomics of VEN + AraC-residual cell populations revealed adaptive resistance associated with changes in oxidative phosphorylation, electron transport chain complex and the TP53 pathway. Accordingly, treatment of VEN + AraC-resistant AML cells with electron transport chain complex inhibitors, pyruvate dehydrogenase inhibitors or mitochondrial ClpP protease agonists substantially delayed relapse following VEN + AraC. These findings highlight the central role of mitochondrial adaptation during AML therapy and provide a scientific rationale for alternating VEN + azacytidine with VEN + AraC in patients with a high MitoScore and to target mitochondrial metabolism to enhance the sensitivity of AML cells to currently approved therapies.