Target Acute Myeloid Leukemia Cells Research Articles

Bispecific Anti-CD123 x Anti-CD3 Adaptir™ Molecules APVO436 and APVO437 Have Broad Activity Against Primary Human AML Cells In Vitro

Background: The IL-3 receptor CD123 is an attractive target for antibody-based therapies in acute myeloid leukemia (AML) because of its frequent expression on AML blasts and its reported overexpression on leukemic stem cells (LSCs) as compared to normal hematopoietic tissues. Previously, two CD123 x CD3 bispecific targeting ADAPTIR constructs (APVO436 and APVO437) have been shown to bind CD123 with high affinity, engage T-cells and, in the presence of effector T-cells, cause cytotoxicity in CD123-expressing cell lines as well as in an in vivo xenograft model. Here, we further investigated the determinants of anti-tumor efficacy of these constructs in human AML.Materials and Methods: Frozen aliquots of Ficoll-isolated mononuclear cells from peripheral blood or bone marrow specimens were obtained from adults with AML. CD123 expression on myeloblasts and cells lines and the percentage of endogenous CD3+ T-cells in AML specimens were quantified by flow cytometry. To determine specific cytotoxicity, cell lines or primary AML cells were incubated in culture medium containing various concentrations of ADAPTIR constructs APVO436 and APVO437. Exogenous T-cells isolated from healthy volunteers were labeled with CellVue membrane dye and added at different effector:target (E:T) cell ratios. After 48 hours, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined by flow cytometry; target AML cells were identified by forward/side scatter properties and negativity for CellVue dye. Specific cytotoxicity was calculated as: 100 x (%DAPItreated - %DAPIcontrol)/(100-%DAPIcontrol) and are presented as mean±SEM of all samples tested. T-cell activation, proliferation and depletion of CD123+ cell populations in normal and AML specimens were assessed using multi-color flow cytometry.Results: We examined the ability of APVO436 and APVO437 to induce cytotoxicity in primary human AML specimens. Of 20 samples tested, 10 had both >60% viability on thaw and >40% viability after 48 hours in cultures and were included for further analysis. The median percentage of myeloid blasts and CD3+ T-cells in the analyzed specimens was 78.5% (range 52-91.7%) and 2.95% (range 0.3-8.8%). With no exogenous T-cells, APVO436 and APVO437 induced modest cytotoxicity; at 1000 pM, APVO436 and APVO437 induced specific cytotoxicity of 6.8±3.8% and 3.9±3.6%, respectively. There was no statistically significant correlation between the percentage of endogenous T-cells present in the sample and the amount of cytotoxicity in the absence of exogenous T-cells. The addition of healthy donor T-cells increased cytotoxicity in a E:T-dependent manner (p <0.0001 for both APVO436 and APVO437) and increasing drug concentrations also increased cytotoxicity in a statistically-significant fashion (p <0.0001 for both APVO436 and APVO437). There was no consistent linear relationship between MFICD123 of blasts (MFICD123, arbitrary units) and cytotoxicity. Significant cytotoxicity was achieved even at the lowest levels of blast CD123 expression (MFICD123 range among patient samples: 349-3423). Inclusion of all 20 samples in cytotoxicity analysis did not qualitatively change results. APVO436 induced T-cell activation in both normal and AML samples at 24 hours as assessed by quantification of CD25 and CD69 expressing T-cells. T-cell activation was accompanied by proliferation of T-cells in normal and AML specimens at 96 hours in the presence of APVO436. APVO436 induced dose dependent depletion of CD123+ cells which was evident at 24 hours in both normal and AML samples.Conclusion: APVO436 and APVO437 cause target cell cytolysis in a dose-dependent, target-antigen-dependent, and E:T-dependent manner. Broad activity was seen in primary human AML samples and both constructs were effective in inducing cytotoxicity across a range of blast CD123 expression levels, including specimens with very limited CD123 expression. Endogenous T-cell activation and proliferation accompanied by CD123+ cell depletion demonstrate similar activity of anti-CD123 x anti-CD3 ADAPTIRs in both normal and AML subject samples. These data are supportive of further investigation of anti-CD123 x anti-CD3 ADAPTIR molecules as a potential treatment option for AML. DisclosuresGottschalk:Aptevo Therapeutics: Employment, Equity Ownership. Comeau:Aptevo Therapeutics: Employment, Equity Ownership. Hoyos:Aptevo Therapeutics: Employment, Equity Ownership. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding.

Antineoplastic effect of a diphtheria toxin-based nanoparticle targeting acute myeloid leukemia cells overexpressing CXCR4

Nanomedicine has opened an opportunity to improve current clinical practice by enhancing the selectivity in the delivery of antitumor drugs to specific cancer cells. These new strategies are able to bypass toxicity on normal cells increasing the effectiveness of current anticancer treatments. In acute myeloid leukemia (AML) current chemotherapy treatments generate a relevant toxic impact in normal cells and severe side effects or even patient death. In this study, we have designed a self-assembling protein nanoparticle, T22-DITOX-H6, which incorporates a ligand (T22) targeting CXCR4-overexpressing (CXCR4+) cells, and a potent cytotoxic diphtheria toxin domain. CXCR4 is overexpressed in AML leukemic cells and associates with poor prognosis, being, therefore, a relevant clinical target. We demonstrate here that T22-DITOX-H6 induces apoptosis in CXCR4+ leukemic cells through CXCR4-dependent internalization. In addition, repeated T22-DITOX-H6 treatment (10 μg/dose per 10 doses, intravenously injected) in a disseminated AML mouse model (NSG mice intravenously injected with THP-1-Luci cells, n = 10 per group) potently blocks the dissemination of AML cells in bone marrow, spleen and liver of treated mice, without inducing toxicity in healthy tissues. In conclusion, our strategy of selectively ablating CXCR4 positive leukemic cells by administering the T22-DITOX-H6 nanoparticle could be a promising treatment, especially in patients undergoing AML relapse after chemotherapy, in which leukemic cells overexpress CXCR4.

A First-in-Human Phase I/IIa Study of Allogeneic Double Negative T Cell for the Treatment of Relapsed AML Post Allo-HSCT

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for most of acute myeloid leukemia (AML) patients. But relapse remains the leading cause of mortality after transplant. Ex vivo expanded allogeneic CD4- and CD8- double negative T cells (allo-DNTs) effectively target AML cells without attacking normal tissues in preclinical models. In this first-in-human phase I/IIa trial, twelve patients with relapsed AML after allo-HSCT were treated with allo-DNTs. Nine patients had hematological relapse, 2 with molecular relapse, and 1 had both molecular and extramedullary relapse. The median times from transplant to relapse and relapse to DNT therapy were 10 months (3-18 months) and 4.7 months (1.75-11.7 months), respectively. At the time of DNT-treatment, 11/12 patients had active or residual disease. After lymphodepleting preconditioning, patients received allo-DNTs in three dose cohorts (5x107-4x108/kg). None of the patients developed graft-versus-host disease or > grade 2 DNT treatment-related toxicity; hence, the maximum tolerated dose was not reached. One patient withdrew from this trial. The initial response was observed in 7 out of 11 patients, where 4 patients achieved complete remission (CR), 2 achieved CR with incomplete hematologic recovery, and 1 achieved partial remission. Five patients remain alive and 4 are in continuing CR 374 to 609 days after DNT-treatment. The predicted 1-year overall survival after DNT treatment is 45.5% (95% CI, 16.7%-70.7%). Collectively, the study supports the feasibility, safety, and potential efficacy of allo-DNTs as a new treatment option for patients with relapsing/relapsed AML after allo-HSCT. Trial Registration: The trial was registered on the Chinese Clinical Trial Registry (ChiCTR-IPR-1900022795). Funding: This work was supported by the National Natural Science Foundation of China (grant # 81670165), International Cooperation Projects in Anhui Province (grant # 1804b06020352), the Fundamental Research Funds for the Central Universities (grants # WK9110000001 and WK9110000060), the Canadian Cancer Society Impact Grant (grant # 704121), Canadian Institutes of Health Research (grant # 419699), the Leukemia & Lymphoma Society (grant #R6509-18) and WYZE Biotech Inc. Declaration of Interest: LZ is a co-inventor on several DNT cell technology related patents and intellectual properties (IPs), received a grant and consulting fees from WYZE Biotech Co. Ltd. WYZE Biotech Co. Ltd licensed the Chinese right for one of the patents; and LZ owns shares in WYZE Biotech Co. Ltd.. LY, JL, and ADS are co-inventor on DNT cell technology related patents/IPs. LY, ZX, MW and PT own shares in WYZE Biotech Co., Ltd and are employees of WYZE Biotech Co., Ltd. MISSING REMAINING AUTHORS Ethical Approval: This study protocol was approved by Chinese Ethics Committee of Registering Clinical Trials. Written informed consent was obtained from all the patients or guardians in accordance with the Declaration of Helsinki.

Fe3O4@Pt nanozymes combining with CXCR4 antagonists to synergistically treat acute myeloid leukemia

Abstract Recurrence and drug resistance of acute myeloid leukemia (AML) remained the leading causes of death in blood cancers. The interaction of chemokine receptor 4/chemokine ligand 12 (CXCR4/CXCL12) leads to the trace residue of AML cells in patients after traditional chemotherapy, which hidden security danger for subsequent recurrence. The toxic and side effects of conventional chemotherapy on normal tissues also severely limited the clinical therapeutic efficacy of AML. To overcome these problems, a multifunctional nanoplatform of Fe3O4@Pt composite nanozyme conjugating CXCR4 antagonist was designed, aiming to synergistically treat AML for the first time, in which, the CXCR4 antagonist was used to specifically target AML cells as well as to significantly interfere CXCR4/CXCL12 axis. In the mildly acidic lysosome microenvironment, highly toxic reactive oxygen species (ROS) was generated through the sequential catalytic reactions of Fe3O4@Pt to trigger AML cells apoptosis, leaving the normal cells unharmed. This nanoplatform exhibited a superior synergistic therapeutic efficacy against AML in vitro and in vivo, preventing AML cells from homing to bone marrow and migrating to spleen, lung and liver, which in turn prolonged the survival period of AML mice. Therefore, this designed nanoplatform holds potentials for clinic applications in AML treatment.

Anticancer efficacy of 3-(4-isopropyl) benzylidene-8-ethoxy, 6-methyl, chroman-4-one (SBL-060), a novel, dual, estrogen receptor-Akt kinase inhibitor in acute myeloid leukemia cells.

Estrogen receptor (ER) α is expressed in a subset of patient-derived acute myeloid leukemia (AML) cells, whereas Akt is predominantly expressed in most types of AML. Targeting AML with dual inhibitors is a novel approach to combat the disease. Herein, we examined a novel small molecule, 3-(4-isopropyl) benzylidene-8-ethoxy,6-methyl, chroman-4-one (SBL-060), capable of targeting AML cells by inhibiting ERα and Akt kinase. The chemical properties of SBL-060 were identified by proton nuclear magnetic resonance (1H-NMR), 13C-NMR, and mass spectroscopy. In silico docking was performed using an automated protocol with AutoDock-VINA. THP-1 and HL-60 cell lines were differentiated using phorbol 12-myristate 13-acetate. ERα inhibition was assessed using ELISA. The MTT assay assessed cell viability. Flow cytometry was performed for cell cycle, apoptosis, and p-Akt analyses. Chemical analysis identified the compound as 3-(4-isopropyl) benzylidene-8-ethoxy,6-methyl, chroman-4-one, which showed high binding efficacy toward ER, with a ΔGbinding score of -7.4 kcal/mol. SBL-060 inhibited ERα, exhibiting IC50 values of 448 and 374.3 nM in THP-1 and HL-60 cells, respectively. Regarding inhibited cell proliferation, GI50 values of SBL-060 were 244.1 and 189.9 nM for THP-1 and HL-60 cells, respectively. In addition, a dose-dependent increase in sub G0/G1 phase cell cycle arrest and total apoptosis was observed after treatment with SBL-060 in both cell types. SBL-060 also dose-dependently increased the p-Akt-positive populations in both THP-1 and HL-60 cells. Our results indicate that SBL-060 has excellent efficacy against differentiated AML cell types by inhibiting ER and Akt kinase, warranting further preclinical evaluations.

Evaluation of the human type 3 adenoviral dodecahedron as a vector to target acute myeloid leukemia

Intensive systemic chemotherapy is the gold standard of acute myeloid leukemia (AML) treatment and is associated with considerable off-target toxicities. Safer and targeted delivery systems are thus urgently needed. In this study, we evaluated a virus-like particle derived from the human type 3 adenovirus, called the adenoviral dodecahedron (Dd) to target AML cells. The vectorization of leukemic cells was proved very effective at nanomolar concentrations in a time- and dose-dependent manner, without vector toxicity. The internalization involved clathrin-mediated energy-dependent endocytosis and strongly correlated with the expression of αVβ3 integrin. The treatment of healthy donor peripheral blood mononuclear cells showed a preferential targeting of monocytes compared to lymphocytes and granulocytes. Similarly, monocytes but also AML blasts were the best-vectorized populations in patients while acute lymphoid leukemia blasts were less efficiently targeted. Importantly, AML leukemic stem cells (LSCs) could be addressed. Finally, Dd reached peripheral monocytes and bone marrow hematopoietic stem and progenitor cells following intravenous injection in mice, without excessive spreading in other organs. These findings reveal Dd as a promising myeloid vector especially for therapeutic purposes in AML blasts, LSCs, and progenitor cells.

Safety and Efficacy of Ex Vivo Expanded Healthy Donor-Derived Double Negative T Cells for the Treatment of AML Relapsed after Allogeneic Stem Cell Transplantation: A First in-Human Phase I/IIa Clinical Trial

BACKGROUND Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the main cause of treatment failure for patients with acute myeloid leukemia (AML). Conventional treatments such as donor lymphocyte infusions (DLI) or secondary allo-HSCT as a monotherapy or in combination with novel agents or immunotherapy showed limited improvements on long-term survival. Our previous studies have shown that a rare subset of CD4- and CD8- double negative T cells (DNTs) expanded from periphery of healthy donors are effective in targeting AML cells in vitro and in xenograft models. Also, allogeneic DNTs can be used as an off-the-shelf adoptive cellular therapy (ACT) for AML. We undertook a first in-human phase I/IIa trial to assess the safety and efficacy of escalating doses of DNTs for the treatment of AML patients relapsed post allo-HSCT. This study protocol was approved by Chinese Ethics Committee of Registering Clinical Trials. Informed consent was obtained in accordance with the Declaration of Helsinki. The trial was registered on the Chinese Clinical Trial Registry (ChiCTR-IPR-1900022795). METHODS From June 2019 to June 2020, 12 patients with relapsed AML after allo-HSCT were enrolled in the trials. Three patients received HLA-matched sibling stem cell transplant, and 9 patients received umbilical cord blood transplantation (UCBT). Hematological relapse was found in 9 cases, and molecular relapsed in 3 cases. None of the patients were able to obtain DLI from previous donors for various reasons. Clinical-grade DNTs from peripheral blood of healthy donors were expanded. After lymphodepleting chemotherapy with fludarabine and cyclophosphamide, each patient received three infusions of HLA-mismatched DNTs expanded from healthy volunteers at escalating doses of 5×107, 1×108, or 2-4×108 per kilogram of body weight at one week interval. Numbers of total DNTs and donor-derived DNTs, inflammatory cytokines levels in vivo were measured according to the study protocol. Post-remission therapy was permitted 14 days after the last DNTs infusion according to treating physician's discretion. RESULTS All surviving patients were followed until August 1st, 2020. At a median follow-up of 4.3 months (range 1.7 to 12.2 months), 1 patient withdrew from the trial after the second DNT infusion for a personal reason. 6/11 patients (54.5%) achieved complete response (CR) with 5 of these patients negative for (45.4%) minimal/measurable residual disease (MRD). 4/11 patients (36.3%) achieved partial remission. Up to the last follow-up, 4/11 patients remain CR. Increase in the levels of serum inflammatory cytokines, including interleukin (IL)-6, TNF-alpha, MCP-1, and MIP-1-beta over baseline were observed without signs of cytokine release syndrome, neurotoxicity, or graft-versus-host disease. The most common adverse events related to DNTs treatment were fever and headache. None of the patients showed &amp;gt;grade 2 adverse event (AE). The maximum tolerated dose was not reached. Donor DNT cell expansion was detected as early as 6 hours after infusion and reached the peak at 24 hours after the 1st infusion in most patients, slightly increased after the 2nd and 3rd infusions, and lasted for 2 weeks after 3rd infusion. However, total DNTs increased and reached the peak at 2 weeks after 3rd infusion. CONCLUSIONS Our results show that allogeneic DNTs therapy is safe, well tolerated, and potentially effective in treating AML patients who relapsed after allo-HSCT. The total number of DNTs increased after infusion. Compared to other agents, DNTs more effectively induced complete responses in this patient population. Our on-going study is to elucidate the mechanism of DNTs-mediated leukemia clearance, and explore the use of DNTs as carrier for Chimeric Antigen Receptor or transgenic TCR to further enhance the efficacy targeting resistant AML cells. Disclosures Zhang: AbbVie: Current equity holder in publicly-traded company; University Health Network: Consultancy, Current equity holder in private company, Other: Principal Investigator, Patents &amp; Royalties.

CXCR4-Expressing Anti-CD25 CAR T-Cells Effectively Eliminate Human AML Cells In Vivo

Chimeric antigen receptor (CAR) T-cells targeting CD19 has become a promising treatment option for relapsed/refractory B cell acute lymphoblastic leukemia and diffuse large B Cell lymphoma. For acute myeloid leukemia (AML), CAR T-cells targeting molecules such as CD33 and CD123 are under clinical evaluation. Regardless of target diseases or antigens, it is essential to understand mechanisms underlying both on- and off-target effects of CAR T-cells such as cytokine release syndrome. In the study, we aimed to develop a CAR T-cell treatment for poor prognosis AML. To this end, we analyzed gene expression of patient-derived AML-initiating cells with demonstrated capacity for in vivo AML development in a NOD/SCID/Il2rgKO (NSG) xenogeneic transplantation assay. CD25 (IL-2 receptor alpha chain), previously reported as a marker for poor prognosis in AML, was over-represented in AML-initiating cells as compared with normal CD34+CD38- hematopoietic stem/progenitor cells (HSPCs) (Saito et al., Science Translational Medicine 2010). In addition, the antigen is expressed in other hematologic malignancies such as CML, adult T cell leukemia/lymphoma, and Hodgkin's lymphoma. We therefore engineered lentiviral vector containing TCR sequence and Fab antigen recognition sites for human CD25 antigen. Following transduction of CD25-CAR lentiviral particles into cord blood-derived human T cells, we achieved in vitro CD25-CAR T-cell expansion to more than 2x107 cells. In vivo treatment of human AML patient-derived xenotransplantation (PDX) mice with 5x106 CD25-CAR (25CAR) T-cells (patient n=3, PDX n=3 for each patient) resulted in reduction of patient-derived leukemic cells in the peripheral blood (PB) of PDX mice, but abundant leukemic cells remained in the bone marrow (BM). To improve homing and targeting of AML cells in the BM, we engineered CAR construct with mouse CXCR4 expression (CXCR4-25CAR). Injection of 5x106 CXCR4-25CAR T-cells resulted in complete elimination of human AML cells in PB of PDX mice (0.0+/-0.0 hCD33+ cells/ml, n=3), while patient-derived AML cells remained in peripheral blood of PDX treated with 5x106 non-CXCR4-expressing 25CAR T-cells (1416.2+/-661.0 hCD33+ cells/ml PB, n=3) (Untreated PDX: 15677 hCD33+ cells/ml PB, n=1). At 4 weeks post-CAR T-cell injection, we found complete eradication of hCD33+ AML cells only in mice treated with CXCR4-25CAR T (CXCR4-25CAR T-cell treated: BM 0.0+/-0.0 cells, spleen 0.0+/-0.0 cells spleen, n=3; 25CAR T-cell treated: BM 1.9+/-0.4x107 cells, spleen 6.2+/-2.8x107 cells, n=3). In addition, histopathological examination demonstrated no xenogeneic GVHD in liver, lung, and intestine of the CXCR4 25 CAR-treated mice. In one CXCR4-25CAR T-cell-treated PDX mouse with longer-term observation, the number of CXCR4-25 CAR T-cells decreased and murine CD45+ hematopoietic cells increased in PB without evidence of AML relapse after 3 months (2 weeks post-injection: hCD33+AML 1040.3 +/-159.7 cells/ml, hCD3+T cells 474.7 +/-85.8 cells/ml, mouse CD45+ cells 751 +/-31.0 cells/ml, n=3; 3 months post-injection: hCD33+AML 0.0+/-0.0 cells/ml, hCD3+T cells 136.3 +/-77.6 cells/ml, mCD45+ cells 1864 +/-428 cells/ml, n=3). These findings indicate that elimination of human AML cells is mediated by specific targeting of CD25 by CAR T-cells. Injection of CXCR4-expressing CD25-CAR T-cells in NSG mice engrafted with normal CB CD34+CD38- HPSCs resulted in no change in the percentage of human CD3+Foxp3+ cells among PB CD4+ T cells (Pre-injection 10.5+/-1.4%, n=2; 4 weeks post-injection: 14.8+/-0.9% n=2). Additionally, CXCR4-25CAR did not affect to cell numbers of CD3+CD4+FoxP3+ cells, total CD3+ cells, CD19+ cells, CD33+ cells, and CD56+ cells in PB, BM, and spleen. CXCR4-expressing CD25-CAR T-cells is a promising treatment strategy for poor prognosis AML. Disclosures No relevant conflicts of interest to declare.

Targeting AXL Kinase Sensitizes Acute Myeloid Leukemia Stem and Progenitor Cells to Venetoclax Treatment

Acute myeloid leukemia (AML) is a heterogeneous haematological cancer characterized phenotypically by the rapid clonal growth of myeloid cells and an accumulation of blasts in the peripheral blood and bone marrow of patients. Despite the major progress that has been made in categorizing different genetic and molecular AML subgroups, therapies and long-term patient outcomes have not changed significantly over the past four decades. Recently, venetoclax (ABT-199), a BH3-mimetic and selective BCL-2 inhibitor, was approved for the treatment of older patients with AML. However, the limited efficacy, drug resistance in complex karyotype AML and disease progression on venetoclax as well as the inherent resistance of leukemic stem cells (LSCs) to therapy pose significant clinical challenges, warranting identification of novel targets and improved treatment strategies. One candidate target is AXL, a member of the TYRO3/AXL/MER (TAM) family of receptor tyrosine kinases. AXL and its ligand growth arrest-specific gene 6 (GAS6) are elevated in AML patients and LSCs, and associated with poor prognosis. To test whether targeting of the AXL/GAS6 pathway is a feasible treatment strategy for AML, in particular to eradicate LSCs, we developed SLC-391, a novel, potent and selective AXL inhibitor. In vitro and in vivo evaluations of the pharmaceutical properties of SLC-391 indicated reasonable solubility, excellent metabolic stability as well as desirable bioavailability in mice and rats. In silico molecular docking analysis showed that SLC391 can adopt a conformation with surface and charge complementary to the active site of the AXL kinase, potentially engaging in hydrophobic ring-mediated interactions. Further, cell-based studies discovered that SLC-391 targets AML cells with high AXL/GAS6 expression, particularly MLL+ AML cells, and synergizes with venetoclax in cell viability and apoptosis assays (CI&amp;lt;0.6). In addition, simultaneous AXL and BCL-2 inhibition reduced the clonal short- and long-term growth of primitive AML patient cells in CFC re-plating and LTC-IC assays compared to single or control treatments (20-95% inhibition). Moreover, a combination of AXL inhibition and venetoclax treatment was able to target LSCs and AML blasts in two different preclinical patient-derived xenotransplantation (PDX) models, extending the mean survival of these mice by 14-30 days compared to single agents (P&amp;lt;0.025). Mechanistically, single-cell RNA-sequencing and functional validation studies revealed that AXL inhibition perturbs oxidative metabolism, and differentially targets signaling pathways to synergize with venetoclax in leukemic cell killing. Importantly, the combination of AXL inhibition plus venetoclax treatment was not toxic to normal BM cells from healthy donors. Hence, our findings identify a promising, improved and specific treatment strategy for AML, particularly patients with high AXL/GAS6 expression. Disclosures No relevant conflicts of interest to declare.

Flotetuzumab and Other Cellular Immunotherapies Upregulate MHC Class II Expression on Acute Myeloid Leukemia Cells in Vitro and In Vivo

Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative therapy for patients with high-risk and refractory acute myeloid leukemia (AML). Unfortunately, up to 50 percent of patients relapse after allo-HCT.Recent research has shown that 30-50 percent of AML samples from patients relapsing after allo-HCT have downregulation of MHC class II (MHC-II) expression, which may promote immune effector evasion and disease relapse. These studies also report that interferon gamma (IFNγ) can restore MHC-II expression. IFNγ has never been systemically administered after allo-HCT and would likely cause significant and potentially life-threatening toxicities. Reinduction of MHC-II expression may lead to re-engagement of immune effectors, restoration of the graft-versus-malignancy effect, and disease control. We hypothesized that T cell immunotherapies targeting AML cells will lead to T cell activation, localized IFNγ release, and upregulation of MHC-II on AML cells. Methods: For in vitro experiments, THP1 cells (THP1s), which have intermediate MHC-II expression, or primary human AML samples with low MHC-II expression from a patient relapsing after allo-HCT (AML-low cells) were co-cultured with or without T-cell immunotherapy and with or without human MHC-mismatched CD3+ T cells. The following T-cell immunotherapies were tested: flotetuzumab (FLZ), an investigational CD123 x CD3 bispecific DART® molecule; a CD33 x CD3 bispecific molecule (Creative Biolabs, Shirley, NY); and CD123-directed chimeric antigen receptor (CAR) T cells. THP1 IFNγ receptor-1 (IFNγR1) knockout cell lines were generated using CRISPR-Cas9. MHC-II expression was measured by flow cytometry and IFNγ concentrations via Luminex immunoflourescence assay. In order to rescue THP1s from FLZ-induced death and allow for longitudinal evaluation, a transwell plate system was used, placing THP1s, human CD3+ T cells, and FLZ in the top well and THP1s in the bottom well. This allowed for diffusion of IFNγ but not human T cells to the bottom wells, permitting MHC-II upregulation while limiting death. The upper and lower wells were coincubated together for 24 hours prior to harvesting of the THP1s in the lower well for longitudinal studies and mixed-lymphocyte reactions. For in vivo experiments, NOD-scid IL2Rgammanull mice expressing human IL-3, GM-CSF, and SCF (NSG-S) were irradiated with 250 rads and injected with 10e6 primary AML-low cells per mouse. After 5.5 weeks, mice were divided into the following groups: 1) untreated control; 2) FLZ only (2mg/kg); 3) human mismatched T cells only (10e7 T cells per mouse); 4) FLZ and T cells. Results: In vitro co-culture of THP1 or AML-low cells with FLZ and T cells led to significantly increased MHC-II expression at 48 hours when compared with the control, FLZ only, and T cell only groups (Figure 1A-B). Co-culture of THP1s with the CD123 CAR-T cells led to similar results. Although co-incubation with a CD33 x CD3 bispecific led to a similar result, the MHC-II upregulation was not nearly as dramatic as that seen with CD123 targeting agents. Using a transwell system to rescue THP1s from FLZ-mediated toxicity, FLZ-induced MHC-II upregulation on THP1s peaked at 48-72 hours (similar kinetics to what is seen with IFNγ alone). These THP1s with upregulated MHC-II activated third-party donor mismatched human CD4+ T cells to a greater extent than untreated THP1s controls. Co-cultures of THP1s with CD4+ T cells and FLZ induced the secretion of very high concentrations of IFNγ, and blockade of IFNγ signaling through knockout of IFNγR1 led to abrogation of the effect (Figure 1C-D). Finally, in an in vivo model, NSG-S mice injected with AML-low samples and treated with FLZ and T cells showed significant upregulation of MHC-II expression on the AML cells. Single cell RNA-sequencing of AML cells purified from these mice is ongoing. Conclusions: Use of FLZ and other T-cell immunotherapies targeting AML antigens led to both direct AML killing as well as significant upregulation of MHC-II expression on AML cells both in vitro and in vivo. The effect appears to be mediated primarily by IFNγ. T-cell immunotherapies represent a promising treatment approach for AML patients relapsing after allo-HCT and may lead to enhanced immune recognition in the 30-50% of patients who relapse after allo-HCT. Based on these results, a clinical trial treating patients relapsing after allo-HCT with FLZ is planned. Disclosures Christopher: Boulder Bioscience: Patents &amp; Royalties: IP around the use of interferon gamma to treat stem cell transplant. Kim:Tmunity: Patents &amp; Royalties: methods for gene editing in hematopoietic stem cells to enhance the therapeutic efficacy of antigen-specific immunotherapy (Licensed by University of Pennsylvania); Neoimmune Tech: Patents &amp; Royalties: use of long-acting IL-7 analogs to enhance CAR T cells (licensed by Washington University). Muth:MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Davidson:MacroGenics: Current Employment. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Abstract A30: Targeting of the mTOR pathway in human acute myeloid leukemia cells using functionalized gold nanoparticles

Abstract Specific targeting of fundamental biochemical/signaling pathways in human malignant cells with the purpose of recognition and delivery of cytotoxic drugs into them is a very promising but not well-developed strategic approach. It is a major focus of current molecular cancer research, nanomedicine, biotechnology, and synthetic biology. mTOR pathway is a master regulator of metabolism and cell growth in the majority of malignant cells. Especially this applies to cancerous myeloid cells where it determines the ability of the cells to survive and evade the host immune surveillance. Therefore, specific targeting of this pathway could be a novel strategy for treatment of acute myeloid leukemia (AML), a blood/bone marrow cancer that can rapidly progress into a systemic and often fatal malignancy. In this study we demonstrated for the first time a fundamentally novel approach for specific targeting of human AML cells by functionalized gold nanoparticles. The immune receptor Tim-3 is highly expressed in human AML cells. It is one of the key members of the Tim-3/galectin-9 autocrine pathway that plays a pivotal role in AML cell survival and immune evasion (1). Rapamycin inhibits the mTOR activity, thus affecting translational pathways in AML cells. Inhibition of the mTOR leads to a rapid killing of AML cells. Using these nanomaterials, we managed to successfully deliver rapamycin into AML cells, reaching attenuation of the mTOR activity. Concentration of rapamycin required to reach such a strong effect was at least 50 times lower compared to that of free rapamycin required to achieve the similar result. We therefore concluded that our technology is of potential use for specific targeting of AML cells for the purpose of diagnosis and possibly therapy (2). The nanoconjugates developed in our work can be used to specifically identify malignant blood cells, thus allowing rapid and robust AML diagnosis. Citation Format: Inna M. Yasinska, Bernhard F. Gibbs, Luca Varani, Rohanah Hussain, Giuliano Siligardi, Elizaveta Fasler-Kan, Luigi Calzolai, Vadim V. Sumbayev. Targeting of the mTOR pathway in human acute myeloid leukemia cells using functionalized gold nanoparticles [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A30.

Panel-based next-generation sequencing facilitates the characterization of childhood acute myeloid leukemia in clinical settings.

Acute myeloid leukemia (AML) accounts for ~20% of pediatric leukemia cases. The prognosis of pediatric AML has been improved in recent decades, but it trails that of most other types of pediatric cancer, with mortality rates of 30-40%. Consequently, newer more targeted drugs are required for incorporation into treatment plans. These newer drugs selectively target AML cells with specific gene alterations. However, there are significant differences in genetic alterations between adult and pediatric patients with AML. In the present study, inexpensive and rapid next-generation sequencing (NGS) of >150 cancer-related genes was performed for matched diagnostic, remission and relapse (if any) samples from 27 pediatric patients with AML. In this analysis, seven genes were recurrently mutated. KRAS was mutated in seven patients, NRAS was mutated in three patients, and KIT, GATA1, WT1, PTPN11, JAK3 and FLT3 were each mutated in two patients. Among patients with relapsed AML, six harbored KRAS mutations at diagnosis; however, four of these patients lost these mutations at relapse. Additionally, two genetic alterations (FLT3-ITD and TP53 alterations) were detected among patients who eventually relapsed, and these mutations are reported to be adverse prognostic factors for adult patients with AML. This panel-based, targeted sequencing approach may be useful in determining the genetic background of pediatric AML and improving the prediction of treatment response and detection of potentially targetable gene alterations. RAS pathway mutations were highly unstable at relapse; therefore, these mutations should be chosen as a target with caution. Incorporating this panel-based NGS approach into the clinical setting may allow for a patient-oriented strategy of precision treatment for childhood AML.

Abstract 5662: JNJ-67571244: A novel anti-CD33 C2 domain binding bispecific antibody with potent T cell redirection activity

Abstract CD33 is expressed in 90% of acute myeloid leukemia (AML) patients with expression in both blasts and leukemic stem cells. The extracellular portion of CD33 consists of a V and a C2 Ig-like domain. A recent study showed that a single nucleotide polymorphism (SNP) rs12459419 (C &amp;gt;T; Ala14Val in exon 2 of CD33) was present in ~50% of the Western AML population and is associated with preferential expression of an alternatively spliced CD33 isoform lacking exon2, resulting in the deletion of the CD33 V domain. Interestingly, several CD33-antibody-based therapies, including gemtuzumab ozogamicin (GO), the only approved anti-CD33 antibody drug conjugate for AML, bind and recognize the V domain of CD33. Recent data demonstrated that patients with SNP rs12459419 CC genotype receiving GO had a significantly lower risk of relapse and increased event-free survival compared to patients with the CT or TT genotypes. Given the data with GO, it is reasonable to hypothesize that the activity of other V binding CD33 antibodies maybe limited to a subset (~50%, rs12459419 CC genotype) of AML patients. On the other hand, since the C2 domain is shared by all CD33 isoforms in AML patients, we hypothesized that a C2 binding anti-CD33 antibody could target AML cells more broadly regardless of their SNP status. Additionally, we reasoned that targeting the membrane proximal C2 domain would be a beneficial strategy for a CD3 redirection bispecific antibody as targeting epitopes closer to the membrane have been reported to mediate efficient synapse formation between T cells and target cells leading to potent anti-tumor responses. We developed JNJ-67571244, a novel human bispecific antibody capable of binding to the C2 domain of CD33, and to CD3 to induce T-cell recruitment and tumor cell cytotoxicity independently of their SNP status. JNJ-67571244 specifically binds to CD33-expressing target cells and induces cytotoxicity of CD33+ AML cell lines in vitro at 48 hours (EC50 values: KG-1=0.168 nM, MOLM-13=0.131 nM, Kasumi-1=0.05 nM and OCI-AML3=0.183 nM) with concomitant T cell activation (EC50 values: KG-1=0.066 nM, MOLM-13=0.028 nM, Kasumi-1=0.043 nM and OCI-AML3=0.05 nM) along with cytokine release. In contrast, JNJ-67571244 was unable to kill CD33- cancer cell lines (CARNAVAL and KG-1 cells with a genetic deletion of CD33), demonstrating the specificity of the cytotoxicity. JNJ-67571244 demonstrated statistically significant anti-tumor activity in vivo in established disseminated and subcutaneous mouse models of human AML (MOLM-13Luc and KG-1: up to 100% and 92% tumor growth inhibition respectively) through T cell redirection activity. Furthermore, this antibody could deplete CD33+ blasts (EC50=0.549 nM) in AML patient blood samples (n=7) in an ex-vivo assay at 48 hours with concurrent T cell activation (EC50=0.355 nM). JNJ-67571244 also cross-reacts with cyno CD33 and CD3 and was well-tolerated in cynomolgus monkeys up to 30 mg/kg along with a sustained reduction in CD33+ leukocyte populations. Lastly, JNJ-67571244 mediated efficient cytotoxicity of cell lines and primary samples regardless of their genotype status (SNP rs12459419 CC, CT and TT), suggesting a potential therapeutic advantage over competitor V-binding antibodies. JNJ-67571244 is currently in Phase 1 clinical trials to treat relapsed/refractory AML and high risk myelodysplastic syndrome (MDS) patients (NCT03915379). Citation Format: Priyanka Nair-Gupta, Michael Diem, Dara Reeves, Weirong Wang, Robert Schulingkamp, Katrin Sproesser, Bethany Mattson, Bradley Heidrich, Jocelin Joseph, Jocelyn Sendecki, Brad Foulk, Gerald Chu, Damien Fink, Qun Jiao, Sheng-Jiun Wu, Kathryn Packman, Yusri Elsayed, Ricardo Attar, Francois Gaudet. JNJ-67571244: A novel anti-CD33 C2 domain binding bispecific antibody with potent T cell redirection activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5662.

Immune escape and immunotherapy of acute myeloid leukemia.

In spite of the recent approval of new promising targeted therapies, the clinical outcome of patients with acute myeloid leukemia (AML) remains suboptimal, prompting the search for additional and synergistic therapeutic rationales. It is increasingly evident that the bone marrow immune environment of AML patients is profoundly altered, contributing to the severity of the disease but also providing several windows of opportunity to prompt or rewire a proficient antitumor immune surveillance. In this Review, we present current evidence on immune defects in AML, discuss the challenges with selective targeting of AML cells, and summarize the clinical results and immunologic insights from studies that are testing the latest immunotherapy approaches to specifically target AML cells (antibodies, cellular therapies) or more broadly reactivate antileukemia immunity (vaccines, checkpoint blockade). Given the complex interactions between AML cells and the many components of their environment, it is reasonable to surmise that the future of immunotherapy in AML lies in the rational combination of complementary immunotherapeutic strategies with chemotherapeutics or other oncogenic pathway inhibitors. Identifying reliable biomarkers of response to improve patient selection and avoid toxicities will be critical in this process.

Somatostatin receptor mediated targeting of acute myeloid leukemia by photodynamic metal complexes for light induced apoptosis

Acute myeloid leukemia (AML) is characterized by relapse and treatment resistance in a major fraction of patients, underlining the need of innovative AML targeting therapies. Here we analysed the therapeutic potential of an innovative biohybrid consisting of the tumor-associated peptide somatostatin and the photosensitizer ruthenium in AML cell lines and primary AML patient samples. Selective toxicity was analyzed by using CD34 enriched cord blood cells as control. Treatment of OCI AML3, HL60 and THP1 resulted in a 92, and 99 and 97% decrease in clonogenic growth compared to the controls. Primary AML cells demonstrated a major response with a 74 to 99% reduction in clonogenicity in 5 of 6 patient samples. In contrast, treatment of CD34+ CB cells resulted in substantially less reduction in colony numbers. Subcellular localization assays of RU-SST in OCI-AML3 cells confirmed strong co-localization of RU-SST in the lysosomes compared to the other cellular organelles. Our data demonstrate that conjugation of a Ruthenium complex with somatostatin is efficiently eradicating LSC candidates of patients with AML. This indicates that receptor mediated lysosomal accumulation of photodynamic metal complexes is a highly attractive approach for targeting AML cells.

NKG2D CAR-Expressing Lymphocytes Target Acute Myeloid Leukemia Cells

Background: Acute myeloid leukemia (AML) is a hematological malignancy with a very low overall survival. Among the new treatment modalities, chimeric antigen receptor (CAR) therapy is showing promising results in other hematological malignancies. Since AML exhibits high heterogeneity and does not have specific differential antigens of the hematopoietic stem cell, using NKG2D-CAR cells could be an appropriate therapeutic strategy against AML. NKG2D receptor has a wide range of specific tumor cell ligands (MICA, MICB, ULBP-1, ULBP-2 and ULBP-3) which are expressed in more than 80% of all tumors. For this reason, the objective of this work was to evaluate the anti-tumor activity of activated and expanded natural killer cells (NKAE) and T cells expressing an NKG2D CAR. Methods: T cells and NK cells were isolated from the healthy donor´s peripheral blood mononuclear cells ring (n = 5) by immunomagnetic depletion. NKAE cells were obtained by co-culture with subletally irradiated CSTX002 cells. The purified NKAEs and T cells were transduced with an NKG2D CAR with 4-1BB and CD3z signaling domains. The efficiency of transduction was evaluated by flow cytometry detecting NKG2D expression. Also, the immunoprofiling of surface molecules and the cytotoxicity against primary blasts of AML, as well as the expression of NKG2D ligands in tumor cells (AML cell lines and primary blasts) were analyzed by flow cytometry. The cytotoxicity of untransduced NKAE, CAR-NKAE cells, untransduced T cells and CAR-T cells was evaluated by 4 hour europium release assay. Toxicity on healthy tissue (healthy lung cells and PBMCs from third party) was analyzed in the same way. The safety of NKG2D-CAR transduced cells was evaluated using CGH arrays to detect chromosomal abnormalities. Results: Both the AML cell lines and primary blasts from AML patients showed expression of the MICA/B and ULBPs-1 to 3 ligands. NKG2D ligands expression was highly variable from a cell line to another. However, all the cell lines and samples showed high expression of at least 1 of the 5 analyzed ligands. Four hour europium release assays revealed that untransduced T cells had higher cytotoxicity than untransduced NKAE cells at the same ratio (32: 1) against both OCI-AML-3 cytarabine-resistant cell line (52.7% ± 14.2% vs. 32.5% ± 7.2%) as to the cytarabine-sensitive cell line (56.42% ± 5.6% vs. 50.14 % ± 5.9%). T cells showed a better transduction efficiency than NKAE cells at a multiplicity of infection (MOI) of 5. It was observed that both the CAR-NKAE cells and the CAR-T cells had higher cytotoxicity against these two lines (OCI-AML-3R and OCI-AML-3S) after being transduced with our NKG2D-CAR. However, the antitumor activity of CAR-T cells always remained superior to that of the CAR-NKAE for both OCI-AML-3R (54.26% ± 3.8% vs. 35.3% ± 6.7%) and for OCI-AML-3S (63.36% ± 3.5% vs. 54.3% ± 3.7%) cell lines, with a greater difference compared to drug resistant cells. The antitumor activity of CAR-T cells and CAR-NKAE cells was always superior on the sensitive cell line. The antitumor activity of NKG2D CAR-T cells was evaluated against primary blasts from AML patients (n = 4), observing a nearly complete destruction of the blasts after 24 hours, at a very low target: effector ratio of 4:1. CAR-T cells populations were analyzed by flow cytometry and we found that CAR-T cells were highly positive to CD45RO and reduced expression of CD45RA, on the other hand, they showed high IL-2 production exhibiting a central memory phenotype. Slight toxicity was observed against lung cells in both cell types. However, CAR-T cells exhibited some toxicity on third party PBMCs being null in the case of CAR-NKAE. CGH arrays studies showed no variation in the copy number, resulting from the introduction of the CAR, of genes which their variation has been associated with chromosomal instability. No significant changes associated with transduction process were observed in the surface phenotype of CAR-NKAE cells or CAR-T cells. Conclusions: We have demonstrated that AML cells could be target with an NKG2D-CAR. Primary NKAE cells and T cells can be transduced with an NKG2D-CAR at a very low MOI to enhance their antileukemic activity. CD3+ CAR-T cells are more effective than CAR-NKAE cells. Moreover, CAR-T cells were able to completely destroy AML blasts. Although further studies are needed, these results show the potential of NKG2D-CAR T and NK cell therapy in AML. Disclosures Rio: Rocket Pharmaceuticals: Equity Ownership, Patents &amp; Royalties, Research Funding. Lee:Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties, Research Funding.

Targeting AML: The Development of Two Functionally and Mechanistically Distinct Classes of Cereblon-Mediated Protein Homeostatic Modulators

Targeting disease-relevant proteins by exploiting the cells' very own protein homeostasis machinery is the next generation drug discovery platform that has come center stage for treatment of hematological malignancies. We present our novel and unique Protein Homeostatic Modulators (PHMsTM) - that promote ubiquitination and subsequent proteasomal degradation of known substrates as well as neosubstrates via Cereblon - as therapeutic candidates for acute myeloid leukemia (AML). Specifically, we report the discovery of two functionally and mechanistically distinct classes of Cereblon-mediated PHMsTM from phenotypic screens of our proprietary PHM®library. Unlike classical IMiDs, our oncology candidates, BTX508 and BTX1119 demonstrate significant cytotoxicity with IC50s in low-nanomolar range in a panel of AML cell lines. BTX508 is a purely cytotoxic compound that does not display immune modulatory activity. In contrast, BTX1119 is not only cytotoxic, but is also immunomodulatory in that it inhibits inflammatory cytokines such as IL-1β, IL-6 and TNF-α as well as induces IL-2, an indicator of T cell activation. BTX1119's immune modulatory activity is a 100-fold more potent when compared to Pomalidomide. The distinct functional activities exhibited by BTX508 and BTX1119 is a result of strategically engineered substrate degradation profiles. Importantly, BTX508 and BTX1119 exhibit a large safety window wherein the concentrations at which they target AML cells do not affect normal healthy cells such as liver epithelial cells or lung fibroblasts. To further establish BTX508 as a clinical candidate for AML, we performed in vivo efficacy studies in the MV-4-11 human AML xenograft model using athymic nude mice. A single daily dose of BTX508 results in a significant reduction in tumor volume. BTX508 exhibits significant oral bioavailability, thus making it a promising clinical candidate for treatment of AML as well as other potential hematological malignancies. With regard to BTX1119, we believe that incorporation of both the potent cytotoxic and immunomodulatory properties into a single molecule holds great therapeutic promise; however, to evaluate the synergistic potential of this combination will require use of a humanized mouse model - this work is ongoing. Disclosures No relevant conflicts of interest to declare.

Inhibition of Fatty Acid Metabolism Re-Sensitizes Resistant Leukemia Stem Cells to Venetoclax with Azacitidine

The combination of venetoclax with hypomethylating agents has resulted in highly promising clinical outcomes for acute myeloid leukemia (AML) patients. However, a subset of patients are refractory or develop resistance to venetoclax based regimens, resulting in disease recurrence. The goal of this project was to determine a mechanism to re-sensitize resistant leukemia stem cells (LSCs) to venetoclax with azacitidine (ven/aza) treatment. LSCs are the population of leukemia cells that initiate disease and are not fully eradicated by conventional treatments resulting in disease recurrence. We have previously reported that ven/aza targets LSCs in de novo AML patients by perturbing amino acid uptake resulting in decreased oxidative phosphorylation (OXPHOS). To investigate how some AML patients, develop resistance to ven/aza, we first determined if ven/aza reduced amino acid uptake in primary human AML ven/aza resistant LSCs by stable isotope labeled metabolic flux and mass spectroscopy analysis. Amino acid uptake was significantly reduced in both ven/aza sensitive and resistant LSCs upon ven/aza treatment, indicating that ven/aza is still biologically active in resistant LSCs. Next, we performed gene expression analysis from LSCs isolated from AML patients who were treated with ven/aza, responded, and then either remained in remission or progressed on ven/aza therapy. Gene set enrichment analysis revealed that fatty acid transport was enriched in LSCs isolated from patients who eventually progressed on ven/aza therapy (FDR = 0.0088) (Figure A). We then determined differences in overall fatty acid levels by lipidomics mass spectroscopy analysis in ven/aza sensitive and resistant LSCs. We observed a significant increase in abundance of 20% (6/29) of fatty acids detected in resistant LSCs. To determine if targeting fatty acid transport could re-sensitize resistant LSCs to ven/aza we knocked down genes involved in fatty acid transport including CD36, CPT1A and CPT1C in 4 ven/aza resistant AML specimens and then measured viability and colony-forming potential upon ven/aza treatment (Figure B and C). Knockdown of CD36, CPT1A, or CPT1C in combination with ven/aza treatment significantly decreased both viability and colony forming ability in each of the AML specimens. In addition, knockdown of CPT1A or CPT1C in combination with ven/aza reduced OXPHOS, a known metabolic requirement of LSCs. To perturb fatty acid transport in a therapeutically relevant manner, we treated LSCs isolated from ven/aza resistant patient specimens with a CPT1 inhibitor, etomoxir, as a single agent or in combination with ven/aza, and then measured viability and OXPHOS. The combination but not single agents reduced viability and OXPHOS, consistent with our genetic studies. To determine if ven/aza with etomoxir targeted functional LSCs we treated a primary AML specimen with etomoxir, ven/aza or the combination and measured engraftment into immune deficient mice. Combination therapy significantly reduced engraftment potential compared to ven/aza or etomoxir alone indicating that the combination of ven/aza with etomoxir decreased LSC function (Figure D). To determine if ven/aza with etomoxir could target AML cells in vivo, we treated a primary patient derived xenograft model with ven/aza, etomoxir, or the combination for 2 weeks and measured leukemic burden in the bone marrow (Figure E). The combination reduced leukemic burden more significantly than ven/aza or etomoxir alone. Finally, we measured the consequences of ven/aza, etomoxir, or the combination on normal hematopoietic stem and progenitor cells. Neither single agents nor combination therapy decreased CD34+ cell viability or colony forming ability, indicating that there may be a therapeutic window to targeting these metabolic pathways in AML without harming normal stem cells. Gene expression analysis revealed that CD36, CPT1A, and CPT1C are expressed at significantly higher levels in AML compared to HSCs, which may contribute to this therapeutic window. In conclusion, these data indicate that ven/aza resistance can be overcome by targeting fatty acid transport in LSCs. Furthermore, combining ven/aza with a CPT1 inhibitor such as etomoxir may be a clinically relevant strategy to overcoming ven/aza resistance. Figure Disclosures Pollyea: Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Forty-Seven: Consultancy, Membership on an entity's Board of Directors or advisory committees; Diachii Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The Distinctive Metabolic Environment of the Bone Marrow Niche Drives Leukemia Chemoresistance

Chemoresistance is the difference between remission and cure in cancer patients and is particularly evident in acute myeloid leukemia (AML) where complete remissions are common, but few are cured. Genetic analysis in patients points to residual founder clones persisting through therapy and ultimately causing relapse. However, most of these genetic alterations are currently not amenable to targeted therapies. We hypothesized that residual chemoresistant cells must pass through extreme metabolic challenges when under selection from chemotherapy and surrounded by massive cell death. Our goal was to define the distinctive metabolic profile of AML cells as they pass through that time of extreme selection pressure and determine whether metabolic pathway manipulation would reveal an exploitable vulnerability for therapy. We used in vivo imaging to define the kinetics of leukemic growth, response to chemotherapy and relapse in the mouse. We reasoned that the moment of maximal response immediately following chemotherapy represents a time when the metabolic milieu is maximally hostile and therefore a distinctive selection moment that the cells ultimately causing relapse must pass through. We captured cells at that time and conducted unbiased metabolic profiling, which revealed a metabolite profile that was distinct from AML cells during pre-chemo growth or late post-chemo relapse. Metabolite enrichment analysis revealed hyperactivation of glutamine metabolism during the extreme selection period, with chemoresistant cells showing high levels of glutamine, glutamate and aspartate. Inhibition of glutamine metabolism using the glutamine analog 6-diazo-5-oxo-L-norleucine specifically during the window of maximal selection pressure reduced the number of chemoresistant cells and improved survival in mouse models of AML. Unexpectedly, we found that residual AML cells were not sensitive to inhibition of the enzyme glutaminase, a target currently under clinical investigation for AML. In vivo metabolic tracer analysis instead revealed that chemoresistant AML cells mainly depend on glutamine nitrogen to fuel pyrimidine synthesis. Aspartate, another essential metabolite for pyrimidine synthesis, can be made from glutamine through the Krebs cycle. Interestingly, we detected almost no glutamine carbon entering the Krebs cycle in vivo even though labeled aspartate was found, indicating a different aspartate source. We found that aspartate levels in peripheral blood plasma are very low compared to those in bone marrow plasma, and explored the possibility of a local bone marrow source. Single cell RNA sequencing of bone marrow stromal cells (BMSC) revealed that CXCL12-positive BMSC express high levels of aspartate synthesis genes and transporters, which further increased in the presence of AML cells. In vitro co-cultures showed that BMSC convert glutamine to aspartate, and transfer this to AML cells. Genetic inhibition of aspartate synthesis in BMSC sensitized AML cells to chemotherapy, showing the importance of this metabolic crosstalk. We propose that induction of a timed metabolic collapse targeting AML cells both directly and indirectly through the bone marrow niche can prevent development of chemoresistance and improve treatment outcomes. Figure Disclosures Scadden: Novartis: Other: Sponsored research; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership; Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The new small tyrosine kinase inhibitor ARQ531 targets acute myeloid leukemia cells by disrupting multiple tumor-addicted programs.

Tyrosine kinases have been implicated in promoting tumorigenesis of several human cancers. Exploiting these vulnerabilities has been shown to be an effective anti-tumor strategy as demonstrated for example by the Bruton tyrosine kinase (BTK) inhibitor, ibrutinib, for treatment of various blood cancers. Here we characterize a new multiple kinase inhibitor, ARQ531, and evaluate its mechanism of action in preclinical models of acute myeloid leukemia. Treatment with ARQ531, by producing global signaling pathway deregulation, resulted in impaired cell cycle progression and survival in a large panel of leukemia cell lines and patient-derived tumor cells, regardless of the specific genetic background and/or the presence of bone marrow stromal cells. RNA-sequencing analysis revealed that ARQ531 constrained tumor cell proliferation and survival through BTK and transcriptional program dysregulation, with proteasome-mediated MYB degradation and depletion of shortlived proteins that are crucial for tumor growth and survival, including ERK, MYC and MCL1. Finally, ARQ531 treatment was effective in a patient-derived leukemia mouse model, causing significant impairment of tumor progression and survival, at tolerated doses. These data justify the clinical development of ARQ531 as a promising targeted agent for the treatment of patients with acute myeloid leukemia.