Patient-derived Acute Myeloid Leukemia Research Articles

A novel IgG-based FLT3xCD3 bispecific antibody for the treatment of AML and B-ALL

BackgroundIn lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for...

Unsupervised discovery of dynamic cell phenotypic states from transmitted light movies.

Identification of cell phenotypic states within heterogeneous populations, along with elucidation of their switching dynamics, is a central challenge in modern biology. Conventional single-cell analysis methods typically provide only indirect, static phenotypic readouts. Transmitted light images, on the other hand, provide direct morphological readouts and can be acquired over time to provide a rich data source for dynamic cell phenotypic state identification. Here, we describe an end-to-end deep learning platform, UPSIDE (Unsupervised Phenotypic State IDEntification), for discovering cell states and their dynamics from transmitted light movies. UPSIDE uses the variational auto-encoder architecture to learn latent cell representations, which are then clustered for state identification, decoded for feature interpretation, and linked across movie frames for transition rate inference. Using UPSIDE, we identified distinct blood cell types in a heterogeneous dataset. We then analyzed movies of patient-derived acute myeloid leukemia cells, from which we identified stem-cell associated morphological states as well as the transition rates to and from these states. UPSIDE opens up the use of transmitted light movies for systematic exploration of cell state heterogeneity and dynamics in biology and medicine.

Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells

Disturbance of the dynamic balance between tyrosine phosphorylation and dephosphorylation of signaling molecules, controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is known to lead to the development of cancer. While most approved targeted cancer therapies are tyrosine kinase inhibitors, PTPs have long been stigmatized as undruggable and have only recently gained renewed attention in drug discovery. One PTP target is the Src-homology 2 domain–containing phosphatase 2 (SHP2). SHP2 is implicated in tumor initiation, progression, metastasis, and treatment resistance, primarily because of its role as a signaling nexus of the extracellular signal–regulated kinase pathway, acting upstream of the small GTPase Ras. Efforts to develop small molecules that target SHP2 are ongoing, and several SHP2 allosteric inhibitors are currently in clinical trials for the treatment of solid tumors. However, while the reported allosteric inhibitors are highly effective against cells expressing WT SHP2, none have significant activity against the most frequent oncogenic SHP2 variants that drive leukemogenesis in several juvenile and acute leukemias. Here, we report the discovery of novel furanylbenzamide molecules as inhibitors of both WT and oncogenic SHP2. Importantly, these inhibitors readily cross cell membranes, bind and inhibit SHP2 under physiological conditions, and effectively decrease the growth of cancer cells, including triple-negative breast cancer cells, acute myeloid leukemia cells expressing either WT or oncogenic SHP2, and patient-derived acute myeloid leukemia cells. These novel compounds are effective chemical probes of active SHP2 and may serve as starting points for therapeutics targeting WT or mutant SHP2 in cancer.

Co-Targeting E-Selectin/CXCR4 with GMI-1359 Facilitates AML Stem Cell Mobilization and Protects BM Niches from Anti-Leukemia Therapy

Acute myeloid leukemia (AML) is characterized by the heterogeneous clonal expansion of undifferentiated myeloid cells in the bone marrow (BM). AML cells compete with normal hematopoietic cells and rewire the BM microenvironment into niches that selectively support leukemia stem cells (LSC). The leukemic niche produces soluble factors that facilitate the retention of LSC and provide protection from cytotoxic and targeted agents. The vascular adhesion molecule, E-selectin is expressed on endothelial cells (EC) in the perivascular niche where therapy-resistant AML cells have an increased affinity to E-selectin compared to normal hematopoietic stem cells (HSC) (Winkler et al., 2020). We previously demonstrated (Chang et al., ASH 2020) that E-selectin blockade by the pharmacological antagonist, GMI-1271 (uproleselan; GlycoMimetics, Inc) sensitized therapy-resistant LSC to Bcl-2 targeted therapy. Efficacious eradication of LSC in the BM however requires blocking multiple receptors and/or associated signaling pathways. A more optimal dislodgement of LSC from the BM could be attained by combining an E-selectin antagonism with blockade of the CXCR4/SDF-1α axis. The dual antagonist of E-selectin and CXCR4, GMI-1359 (GlycoMimetics, Inc.), has been tested in a phase 1 clinical trial (NCT02931214). Previously, we showed that GMI-1359 in combination with a FLT3-ITD inhibitor, improved survival in a xenograft model of FLT3-ITD + AML (Zhang et al., 2016). Hence, we hypothesized that co-targeting E-selectin/CXCR4 more efficiently mobilizes AML cells from BM niches and synergizes with the anti-leukemia activity of venetoclax/hypomethylating agent (Ven/HMA).Intra-vital 2-photon imaging and tracking of individual leukemia cells in triple reporter mice (Blood: dextran-TRITC; Host T-cells: DsRed; Host myeloid CD11 cells: EYFP) injected with AML cells carrying a turquoise fluorescent protein reporter gene suggested that dual inhibition of E-selectin/CXCR4 with GMI-1359 significantly enhanced AML cell motility (Fig 1. from 2.2 um/min to 5.4 um/min, p<0.001). Individual cells were dislodged from the niche and traveled long-distance. The combined inhibition of E-selectin and CXCR4 depleted BM leukemia cells in vascular niche areas. In a patient-derived primary AML xenograft (PDX) model (harboring mutations in JAK2 and c-Kit), combinatorial treatment of GMI-1359 with Ven/HMA significantly reduced BM retention of LSC compared to control cohorts or to Ven/HMA alone (p = 0.02 and p=0.003, respectively).In order to better understand how the augmented AML mobilization improves the efficacy of AML therapy, BM cells from PDX mice treated for 2 weeks with GMI-1271, GMI-1359, Ven/HMA, and their combinations were analyzed by single-cell proteomics (CyTOF). Blockade of E-selectin alone or dual E-selectin/CXCR4 inhibition in combination with Ven/HMA diminished levels of E-selectin ligand, mTOR, pFAK, pRb, cMyc, while increasing p21 and cleaved caspase3, which was associated with significant reduction of BM-resident LSC compared to Ven/HMA alone (CD45+34+CD38-CD123+, p= 0.03). AML blasts from the BM of the combinatorial treatment groups showed altered signaling including decreased Ki67, pRb, pNFkB, pPI3K, and E-selectin ligand, and increased levels of cleaved caspase 3. We further found that Ven/HMA significantly diminished CD31+ EC in the BM compared to control cohorts (p= 0.009). However, pharmacological antagonists of E-selectin or E-selectin/CXCR4 protected EC from Ven/HMA-induced detrimental insults through upregulation of survival signaling cascades including pAKT, pERK, pMAPK and decreased eNOS expression in EC compared to Ven/HMA treatment alone. Both EC and MSC were protected by dual inhibition of E-selectin/CXCR4 with GMI-1359. We also observed upregulated pro-survival signaling pathways such as phosphorylation of AKT-MAPK-ERK along with increased Bcl-xL, Bcl-2, and Idu expression in MSC from the GMI-1359 + Ven/HMA treated PDX mice compared to Ven/HMA single treatment cohorts.Collectively, our results provide strong evidence that co-targeting E-selectin/CXCR4 with GMI-1359 profoundly reduces BM retention of LSC as well as protects BM niche component cells from apoptosis induced by targeted therapy, resulting in improving the anti-leukemia activity of Ven/HMA therapy in AML. [Display omitted] DisclosuresFogler: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Carter: Ascentage: Research Funding; Syndax: Research Funding. Andreeff: Oxford Biomedica UK: Research Funding; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Aptose: Consultancy; Glycomimetics: Consultancy; Medicxi: Consultancy; Senti-Bio: Consultancy.

Multi-Antigen Primed T Cells Promote Apoptosis of Acute Myeloid Leukemia (AML)

IntroductionAcute myeloid leukemia (AML), the most common acute leukemia in adults, is characterized by uncontrolled proliferation of immuature myeloid cells. Despite newly approved drugs, AML remains largely incurable due to the persistence of the leukemia stem cell (LSC) population which lie quiescent in the bone marrow niche. Immunotherapy has potential to eradicate LSCs, however, no unique LSC immunophenotype has been identified. Moreover, it is necessary to simultaneously target multiple antigens (Ags) to prevent immune escape and to overcome refractory disease. We present in vitro studies in support of a therapeutic platform capable of targeting multiple intracellular Ags which could meet this challenge. The adoptive transfer of activated T cells primed to engage diverse AML associated epitopes by ex vivo exposure to artificial Ag presenting cells (aAPC) has the potential to eliminate both primary leukemia blasts and LSCs.HypothesisEx vivo enrichment and expansion (E+E) of antigen-educated CD8+ T cells recognizing 5 peptides derived from 3 proteins, Cyclin A1, PRAME and WT1, can selectively identify, engage, and kill AML cell lines or patient-derived (PD) AML blasts in a HLA A*02:01 restricted manner in vitro.Materials and MethodsT cells from the peripheral blood mononuclear cell fraction of a healthy HLA A*02:01 donor were enriched for antigen-educated CD8+/CD4 -T cells. These cells were cultured with nanoparticles decorated with the 5 peptides and a costimulatory protein, resulting in the activation and expansion of those T cells expressing the cognate T cell receptors. These cells are composed of ~97% abT cells, 3% gdT cells and ~13% T scm, 41.5% T cm, 39.5%T em, 6%T emra and 1% T n.ResultsEx vivo expanded educated T cells exhibit target-specific anti-AML activity. T cell mediated cell apoptosis of HLA-matched THP1 cells is dose and time-dependent. At 10:1 effector to target (E:T) ratio, ~28% apoptosis occurred at 24 hrs, while apoptosis was at basal levels when antigen non-educated T cells were used (data not shown). Studies were extended to PD AML cells (Fig. 1A: 012; Fig. 1B: 415) where antigen educated T cells elicited rapid (<16 hrs) and extensive (~90%) apoptosis of target PD AML cells at all E:T ratios examined. Time lapse photography of T cell/PD AML incubations revealed antigen-educated T cells clustered around AML cells (Fig. 2A), a fraction of the latter disappearing over the course of 12 hrs while PD AML cells incubated with non-educated T cells (Fig. 2B) remained viable over 12 hrs. Furthermore, there is little or no T cell movement or clustering in the wells with unprimed, non-active T cells.Release of IFN-γ by educated T cells. T cells (Fig.3A: antigen-educated throughE+E) were incubated at E:T::5:1 for 24 to 48 hrs and IFN-γ in supernatants measured. The fold difference over non-educated T cells incubated with AML cells for the same time is shown and can reach over 5-fold. IFN-γ accumulation was time-dependent where antigen-educated T cells were combined with HLA-A2 matched THP1 or PD AML cells (012, 415, 470). Educated T cells were not active against target cells lacking HLA-A2 (K562) demonstrating HLA restricted killing (Fig. 3B). Additionally, antigen-educated T cells incubated without any target released slightly more IFN-γ than non-educated T cells under similar conditions but AML cells fail to stimulate IFN-γ release from non-educated T cells (data not shown).ConclusionsWe demonstrate HLA restricted cytotoxic activity of antigen-educated T cells against THP1 AML cells and PD AML blasts as shown by flow cytometry and microscopy. Consistent with target cell death, the supernatants from assays with antigen-educated T cells and HLA A*02:01 AML target cells exhibited over 5-fold more IFN-γ than media from assays of non-educated cells under identical conditions. Under these in vitro conditions, PD AML blasts were more readily killed than THP1 cells perhaps due to higher target antigen density (data not shown). These results support the use of multi-antigen-educated T cells for adoptive transfer to treat AML. To investigate the safety and establish the recommended phase II dose, a multi-center Phase I clinical study is underway in relapsed AML post-allo-HCT (NCT 04284228). Future studies will incorporate new antigens to enable broader targeting of a heterogeneous population of AML within and across patients [Display omitted] DisclosuresOelke: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy.

Inhibition of PARP1 Reduces Cell Viability and Increases Markers of DNA Damage in Fanconi Anemia-Mutated Acute Myeloid Leukemia

Introduction:Fanconi Anemia (FA) is a hereditary disorder characterized by deficiencies in DNA damage repair and genome instability with a high propensity for bone marrow failure (BMF) and malignancies such as acute myeloid leukemia (AML). Clinically, FA patients experience greater toxicity than non-FA patients when treated with cytotoxic chemotherapy used for AML treatment, so there is a need for alternative treatments to be developed for FA-mutated AML.Poly (ADP-ribose) polymerase 1 (PARP1) is an important enzyme involved in the recognition and repair of DNA breaks. There has been recent clinical success in treating cancers with defective DNA damage repair with PARP inhibitors, an example of synthetic lethality. Therefore we hypothesize that PARP inhibition (PARPi) is an effective strategy for treating FA-mutated AML. Recent studies have shown that PARP1 is overexpressed in many cancers, including AML, and that higher PARP1 expression is associated with worse patient outcomes. Here, we investigate the anti-tumor effects of a PARP inhibitor, olaparib, on FA-mutated and wild-type (WT) AML cells and investigate the activity of downstream DNA repair pathways in response to PARPi.Methods/Results:To determine the effects of PARPi on AML and FA-mutated AML cells in vitro, we treated four cell lines, one FA-wild type AML line and three patient-derived FA-mutated AML lines, with olaparib for 1, 4, 8, 24, and 48 hours. Preliminary data suggest that olaparib treatment decreases protein expression of both PARP1 and PAR (from activation of PARP) compared to vehicle controls.To evaluate the effect of PARPi on DNA damage in AML we measured γH2AX expression by western blotting and immunofluorescence, and found that, although γH2AX expression was not significantly increased in FA-wild type AML cells, there was a significant increase in γH2AX expression in SB1685 FA-mutated AML cells treated with olaparib compared to controls after 4 hours of treatment (p-value < 0.05).To further evaluate the ability of olaparib to inhibit DNA damage repair, we treated our cells with olaparib and performed single-cell alkaline electrophoresis COMET assay. We found that, while the WT cell line was able to repair its DNA over time (indicated by lower levels of DNA damage after 48 hours of olaparib exposure compared to earlier time points), our FA-mutated AML cell lines had more DNA damage after 48 hours of treatment compared to controls. These data suggest that, while cells proficient in DNA repair are capable of repairing DNA damage even when exposed to PARPi, cells that have mutations in their ability to repair DNA damage are not only less able to repair DNA damage over time but also show increased DNA damage over time when exposed to PARPi.To better understand the effects of this increase in DNA damage, we treated our cells with olaparib and assayed for cell viability over 96 hours. We found that, while WT AML cells did not have significantly decreased cell viability after 96 hours, FA-mutated cell lines trended towards significant decrease in cell viability at 96 hours. These cell lines were also stained with Annexin V to investigate apoptotic activity. Our results indicate that olaparib is able to induce apoptosis in our FA-mutated cells after 24 hours of treatment and that, as treatment continues, the percent of Annexin V-positive cells increases compared to controls.To investigate downstream DNA damage response to PARPi, we treated our cells with olaparib and analyzed the expression of DNA Ligase III, Mre11, XRCC1, and Rad51-enzymes involved in various DNA repair pathways. We found that expression levels of XRCC1 increased over 48 hours in our WT AML cells, suggesting a response to the DNA damaging effects of PARPi. In our FA-mutated SB1685 cells, we found a decrease in XRCC1, DNA Ligase III, and Rad51. The expression levels of these enzymes in the other FA-mutated cell lines were more variable, suggesting that the impact of PARPi on downstream DNA repair pathways may be different across different cell lines.Conclusions:Our data suggest that PARP inhibition may be a potential therapy for the treatment of acute myeloid leukemia. In particular, leukemia with mutations in DNA repair mechanisms may be more responsive to PARP inhibition due to resulting DNA damage and synthetic lethality. Thus, PARP inhibitors have the potential to be an effective therapeutic strategy for the treatment of FA-mutated AML. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Activity of Selective SYK Inhibitors, Entospletinib and Lanraplenib, Alone or Combined with Targeted Agents in Ex Vivo AML Models with Diverse Mutational Backgrounds

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that mediates integrin and Fc receptor signaling in myeloid cells. SYK has been implicated as an oncogenic driver in acute myeloid leukemia (AML) with aberrant expression of HOXA9 and MEIS1 and cooperates with FLT3 internal tandem duplication to drive leukemogenesis. The oral SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity in HOXA9/MEIS1 driven AML and is currently being investigated in a phase 3 trial of previously untreated patients with nucleophosmin1-mutated (NPM1 mut) AML. Lanraplenib (LANRA) is a next generation oral SYK inhibitor with potency and selectivity comparable to ENTO. In healthy volunteers and patients with autoimmune disease, LANRA has shown pharmacokinetic properties that compare favorably with ENTO. To support the clinical development of LANRA for the treatment of AML, ex vivo treatment of patient-derived AML cells was used to compare its activity to that of ENTO, both as a single-agent and in combination with other AML therapies. First, ENTO and LANRA single-agent activities were evaluated in peripheral blood-derived blasts from 15 AML patients, representing different mutational backgrounds including NPM1, FLT3, PTPN11, and NRAS mutations. AML cells were seeded into 96 well plates and treated with ENTO and LANRA for 6 days. Comparable effects on viability were observed across the 15 models with the 2 compounds, and in 11 of the models, the half maximal inhibitory concentration (IC 50) values were within 2-fold of each other. ENTO had a slightly lower IC 50 value than LANRA in the FLT3-mutated models possibly due to the direct FLT3 inhibitory activity of ENTO. Next, we tested the activity of ENTO and LANRA ex vivo in bone marrow-derived AML blasts from 29 AML patients representing diverse mutational backgrounds, including NPM1, IDH1, FLT3, and RAS mutations as well as MLL rearrangements. The models were treated for 9 days with either ENTO or LANRA, and viability was assessed using Annexin V and 7-aminoactinomycin D staining. Again, ENTO and LANRA showed comparable effects on cell viability with no significant differences between the compounds when compared across the different mutational backgrounds. Both studies suggest the potential for anti-leukemic activity in several different genetically defined subsets of AML. Matrix combination assays were performed by combining ENTO or LANRA with either cytarabine (NPM1 mut), gilteritinib (FLT3 mut), or trametinib (RAS mut) with cell viability and death assessed after a 3-day incubation period. Increased cell death in an additive manner was observed in all combinations tested, with results for ENTO and LANRA being similar, indicating the utility of both compounds in combinatorial treatment paradigms. These results support the clinical evaluation of LANRA in genetically defined subsets of AML. A phase 1b/2 study of LANRA in combination with the selective FLT3 inhibitor gilteritinib, in patients with relapsed or refractory FLT3 mut AML is planned for the end of this year. DisclosuresDay: Cyteir Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Heckman: Novartis: Research Funding; Orion Pharma: Research Funding; Celgene/BMS: Research Funding; Oncopeptides: Consultancy, Research Funding; Kronos Bio, Inc.: Research Funding. Schinzel: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Obholzer: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Lin: Kronos Bio, Inc.: Current Employment. Kumar: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. DiMartino: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Saffran: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company.

The Impact of Hypomethylating Agents and BCL-2 Inhibitor on HLA Expression on THP-1 Cells

Note: BP, MV and LG, KG contributed equallyBackgroundRelapsed acute myeloid leukemia (AML) remains the most common reason for allogeneic hematopoietic cell transplant (HCT) failure. Thus, understanding AML immune escape mechanism is important for improving the odds of curing HCT patients with AML. Downregulation of HLA Class I and II expression by AML is one of the potential immune escape mechanisms. Therefore, treatment to restore HLA surface expression is crucial to prevent and treat relapse. Endogenous cytokines, such as IFN-γ, have been shown to stimulate HLA expression but are poorly tolerated by patients. However, two hypomethylating agents (HMA), decitabine (Dec) and azacitadine (Aza), that are routinely used in AML treatment are known to augment HLA expression. For AML, HMAs are often combined with venetoclax (Ven), a drug that blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein. Thus, while HMAs have been reported to increase HLA expression, what is unknown is whether these agents impact individual HLA loci differently and whether Ven has any impact on HLA expression. To address these questions, we treated the THP-1 cell line with Dec, Aza or Ven and measured changes in cell-surface expression of HLA proteins by flow cytometry using locus-specific HLA mAbs.MethodsTHP-1 cells were incubated with IFN-γ (500 U/mL), Aza (2µM), Dec (5µM), or Ven (30nM) for 48 hours (drug concentrations were determined by earlier titration experiments). THP-1 cells are a monocytic cell line, derived from the peripheral blood of a childhood case of acute monocytic leukemia (M5 subtype), that express HLA Class I and HLA-DR but not HLA-DQ or -DP under basal conditions, although they are inducible by IFN-γ. Thus, the induction of HLA Class II expression by IFN-γ serves as a positive control. Isotype controls were included to measure background. Data is presented as the difference in MFI (delta MFI) between cells treated with a drug and those treated with diluent only.ResultsTreatment of THP-1 cells with either IFN-γ or Dec led to increases in Class I HLA-A, -B & -C (Figure 1) compared to untreated cells (a mean fold increase of 1.4 and 1.2, respectively). Notably, Aza did not stimulate additional HLA-C expression and induced less of an increase in HLA-A & -B expression (an increase of 1.1-fold) than IFN-γ or Dec. Treatment of THP-1 cells by Ven did not induce a change in HLA Class I expression.For Class II, IFN-γ or Dec increased HLA-DR, -DQ and -DP expression in comparison to untreated cells (Figure 1). IFN-γ induced greater HLA-DR expression compared to Dec (an increase of 2.3-fold and 1.5-fold, respectively), and both stimulated similar increases in HLA-DQ (increases of 1.5-fold and 1.4-fold, respectively) & -DP (increases of 1.9-fold and 1.5-fold, respectively). However, treatment of cells with either Aza or Ven did not lead to changes in HLA Class II expression.DiscussionPrevious studies have illustrated the ability of IFN-γ to induce HLA Class II expression in THP-1 cells, however, data for Dec to induce HLA Class II expression was unconfirmed. We report differences in the degree to which IFN-γ and Dec are capable of stimulating HLA-DR with IFN-γ being more potent. The inability of Aza to induce HLA Class II expression in THP-1 cells may be related to the differing drug activating pathways of the two HMAs. Indeed, there are conflicting reports as to whether Aza can stimulate HLA Class II expression. Though Ven treatment of THP-1 cells did not impact HLA expression, because it is given with HMAs, it remains to be seen what effect these drugs may have on HLA expression when administered together. Additional studies to confirm these observations in patient-derived AML blasts are ongoing.ConclusionWe report that HMAs increased expression of HLA-A, -B, & -C loci and Dec but not Aza stimulated HLA-DR, -DQ, and -DP expression in THP-1 cells. Given these data, Dec may be superior in increasing HLA Class II expression post-HCT. [Display omitted] DisclosuresMarcucci: Abbvie: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings. Al Malki: Neximmune: Consultancy; CareDx: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; Rigel Pharma: Consultancy; Hansa Biopharma: Consultancy.

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.

Myeloid Leukemia Dependencies at CTCF-Enriched Long Noncoding RNA Loci

The noncoding genome presents a largely untapped source of biological insights, including tens of thousands of long noncoding RNA (lncRNA) loci. While some produce bona fide lncRNAs, others exert transcript-independent cis-regulatory effects, and the lack of predictive features renders their mechanistic dissection highly challenging. Here, we describe CTCF-enriched lncRNA loci (C-LNC) as a putative new subclass of functional genetic elements exemplified by MYNRL15 - myeloid leukemia noncoding regulatory locus on chromosome 15.Initially identified by an expression-guided CRISPRi screen of hematopoietic stem and progenitor (HSPC) / acute myeloid leukemia (AML) lncRNA signatures (480 genes, 1545 sgRNAs), we found MYNRL15 dependency in myeloid leukemia cells of diverse genetic backgrounds. Interestingly, cis and trans perturbation approaches revealed both the MYNRL15 transcript and its flanking protein-coding genes to be dispensable. High density CRISPR tiling of a 15 kb area centered on MYNRL15 (1613 sgRNAs) instead uncovered two crucial, candidate cis-regulatory DNA elements in the locus, which drive the MYNRL15 perturbation phenotype. To determine the molecular basis of MYNRL15 dependence, we performed transcriptome, chromatin conformation, chromatin accessibility, and CTCF profiling. RNA-sequencing established MYNRL15's involvement in maintaining key cancer dependency pathways (e.g. cell cycle, ribosome, spliceosome). Further, MYNRL15 perturbation associated with the coordinated dysregulation of several chromosome 15 neighbourhoods, and formation of a long-range chromatin interaction between the locus and the base of a distal loop, as detected via next-generation Capture-C. The gained interaction was accompanied by diffuse gains in chromatin accessibility across the distal interaction sites (ATAC-seq) as well as reduced CTCF occupancy at the MYRNL15 locus (CTCF CUT&RUN), altogether indicating the 3D re-organization of chromosome 15 following MYNRL15 perturbation. Integrative analysis of the chromatin conformation and transcriptome data, combined with a small CRISPR-Cas9 knockout screen of protein-coding genes from the gained interaction region (29 genes, 149 sgRNAs), pinpointed two potent cancer dependency genes that are located in the region and downregulated following MYNRL15 perturbation: namely, WDR61 and IMP3. Individual knockout of both genes robustly depleted myeloid leukemia cells, recapitulating the MYNRL15 perturbation phenotype and positioning WDR61 and IMP3 as its regulatory targets. Importantly, in primary cells, MYNRL15 perturbation eradicated AML blasts while sparing 50-60% of CD34 + HSPCs in vitro, and reduced patient-derived AML xenografts up to 10-fold in vivo, indicating a potential therapeutic window.Having implicated MYNRL15 in 3D genome organization and demonstrated its role in myeloid leukemia cells, we explored whether MYNRL15 may belong to a sub-category of biologically relevant lncRNA loci that have thus far been overlooked due to their lack of transcript-specific functions. Remarkably, elevated CTCF density (e.g. number of CTCF binding sites per kb of gene length) distinguishes MYNRL15 and 531 other lncRNA loci in K562 cells, of which 43-54% associate with genetic subgroups and/or survival in AML patient cohorts, and 18.4% are functionally required for leukemia maintenance as determined by CRISPR-Cas9 screening. The latter hit identification rate represents a substantial improvement over typical lncRNA essentiality screens (which range from 2-6%) - illustrating the effectiveness of CTCF density metrics in refining functional lncRNA candidate lists, and underlining the relevance such loci hold for AML and cancer pathophysiology in general. Curated C-LNC catalogs in other cell types will facilitate the search for noncoding oncogenic vulnerabilities in AML and other malignancies. [Display omitted] DisclosuresReinhardt: Celgene Corporation: Consultancy; Novartis: Consultancy; Bluebird Bio: Consultancy; Janssen: Consultancy; CLS Behring: Research Funding; Roche: Research Funding. Klusmann: Bluebird Bio: Consultancy; Novartis: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy.

Post-Transplant Vaccination with a Personalized Dendritic Cell/AML Fusion Cell Vaccine for Prevention of Relapse

Post-Transplant Vaccination with a Personalized Dendritic Cell/AML Fusion Cell Vaccine for Prevention of Relapse

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.

FLT3 inhibition by olverembatinib (HQP1351) downregulates MCL-1 and synergizes with BCL-2 inhibitor lisaftoclax (APG-2575) in preclinical models of FLT3-ITD mutant acute myeloid leukemia

IntroductionFLT3-ITD mutations occur in approximately 25% of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Despite initial efficacy, short duration of response and high relapse rates limit clinical use of selective FLT3 inhibitors. Combination approaches with other targeted therapies may achieve better clinical outcomes. Materials and methodsAnti-leukemic activity of multikinase inhibitor olverembatinib (HQP1351), alone or in combination with BCL-2 inhibitor lisaftoclax (APG-2575), was evaluated in FLT3-ITD mutant AML cell lines in vitro and in vivo. A patient-derived FLT3-ITD mutant AML xenograft model was also used to assess the anti-leukemic activity of this combination. ResultsHQP1351 potently induced apoptosis and inhibited FLT3 signaling in FLT3-ITD mutant AML cell lines MV-4-11 and MOLM-13. HQP1351 monotherapy also significantly suppressed growth of FLT3-ITD mutant AML xenograft tumors and prolonged survival of tumor-bearing mice. HQP1351 and APG-2575 synergistically induced apoptosis in FLT3-ITD mutant AML cells and suppressed growth of MV-4–11 xenograft tumors. Combination therapy improved survival of tumor bearing-mice in a systemic MOLM-13 model and showed synergistic anti-leukemic effects in a patient-derived FLT3-ITD mutant AML xenograft model. Mechanistically, HQP1351 downregulated expression of myeloid-cell leukemia 1 (MCL-1) by suppressing FLT3-STAT5 (signal transducer and activator of transcription 5) signaling and thus enhanced APG-2575-induced apoptosis in FLT3-ITD mutant AML cells. ConclusionsFLT3 inhibition by HQP1351 downregulates MCL-1 and synergizes with BCL-2 inhibitor APG-2575 to potentiate cellular apoptosis in FLT3-ITD mutant AML. Our findings provide a scientific rationale for further clinical investigation of HQP1351 combined with APG-2575 in patients with FLT3-ITD mutant AML.

Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy.

Simple SummaryAcute myeloid leukemia (AML) is a type of blood malignancy particularly affecting the myeloid lineage and one of the most common types of leukemia in adults. It is characterized by high heterogeneity among patients leading to immune escape and disease relapse, which challenges the development of immunotherapies such as chimeric antigen receptor (CAR) T-cells. In this way, the aim of our work was to establish the modular RevCAR platform as a combinatorial tumor targeting approach for the treatment of AML. Herein, we demonstrate the preclinical flexibility and efficiency of RevCAR T-cells in targeting patient-derived AML cells expressing CD33 and CD123. Furthermore, AND gate logic targeting these antigens was successfully established using the RevCAR platform. These accomplishments pave the way towards the future clinical translation of such an improved and personalized immunotherapy for AML patients aiming long-lasting anticarcinogenic responses.Clinical translation of novel immunotherapeutic strategies such as chimeric antigen receptor (CAR) T-cells in acute myeloid leukemia (AML) is still at an early stage. Major challenges include immune escape and disease relapse demanding for further improvements in CAR design. To overcome such hurdles, we have invented the switchable, flexible and programmable adaptor Reverse (Rev) CAR platform. This consists of T-cells engineered with RevCARs that are primarily inactive as they express an extracellular short peptide epitope incapable of recognizing surface antigens. RevCAR T-cells can be redirected to tumor antigens and controlled by bispecific antibodies cross-linking RevCAR T- and tumor cells resulting in tumor lysis. Remarkably, the RevCAR platform enables combinatorial tumor targeting following Boolean logic gates. We herein show for the first time the applicability of the RevCAR platform to target myeloid malignancies like AML. Applying in vitro and in vivo models, we have proven that AML cell lines as well as patient-derived AML blasts were efficiently killed by redirected RevCAR T-cells targeting CD33 and CD123 in a flexible manner. Furthermore, by targeting both antigens, a Boolean AND gate logic targeting could be achieved using the RevCAR platform. These accomplishments pave the way towards an improved and personalized immunotherapy for AML patients.

Avocado-derived avocadyne is a potent inhibitor of fatty acid oxidation.

Avocatin-B (Avo-B), an avocado-derived 1:1 mixture of the polyhydroxylated alcohols avocadyne (AYNE) and avocadene, eliminated leukemia cells by suppressing fatty acid oxidation (FAO) in vivo and in vitro while sparing healthy blood cells. In this study, we identified AYNE as the most potent FAO inhibitor within the Avo-B mixture capable of inducing cell death in leukemia cells lines (IC50 : 3.10±0.14µM in TEX cells; 11.53±3.32µM in OCI-AML2) and patient-derived acute myeloid leukemia cells. When added individually, the two Avo-B constituents demonstrated antagonism (Combination Index values >1), highlighting the need for future studies to assess AYNE alone. Together, this work highlighted AYNE as the most potent FAO inhibitor within the Avo-B mixture. PRACTICAL APPLICATIONS: This work identifies which of the two molecules in avocatin B (Avo-B), an avocado-derived mixture of two molecules with demonstrated human safety, utility against leukemia, insulin resistance and diabetes, is most useful. Therefore, it provides the basis for future clinical studies that will focus on testing and developing the most active Avo-B constituent.

Dysregulation of Follistatin Promotes Leukemogenesis in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an abnormal increase of myeloblasts in bone marrow (BM) and peripheral blood (PB). Chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However, these approaches have reached an impasse and despite an initial remission, leukemia relapses are frequent, with an overall cure rate of only 30-40% (Rowe and Tallman 2010). Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene is the commonest mutation in AML, occurring in nearly 30% cases with inferior disease-free and overall survivals (Boissel et al. 2002; Meshinchi et al. 2001). FLT3-ITD as a therapeutic target has been tested in phase I/II clinical trials (Man et al. 2012; Fischer et al. 2010; Levis et al. 2011; Cortes et al. 2011). In most circumstances, there was an initial response to FLT3 inhibitors with reduction or clearance of myeloblasts in BM and PB. However, resistance invariably developed despite continuous treatment (Kindler, Lipka, and Fischer 2010). Therefore, the identification of novel therapeutic targets for FLT3-ITD+ AML becomes urgent.In our previous study (He et al. 2014), we showed that over-expression of human leukemic gene FLT3-ITD resulted in significant myeloid cells expansion which could be effectively rescued by FLT3-ITD inhibitor AC220 treatment. Interestingly, a proportion of zebrafish embryos expressing FLT3-ITD showed double-headed and axis-duplication phenotype as shown by the whole mount in situ hybridization (WISH) of a notochord-specific marker col9a2 . Mechanistically, FLT3-ITD-induced axis-duplication was correlated with the expansion of Spemann's Organizer as shown by the ectopic expression of Spemann's Organizer-specific marker goosecoid (gsc) by WISH as well as by the -2,067gsc-GFP reporter assay. It has been reported that some key signaling pathways are conserved between embryogenesis and tumorigenesis. We hypothesized that certain Spemann's Organizers were dysregulated in FLT3-ITD+ AML.To test this hypothesis, we performed an in silico analysis to quantify the gene expression of Spemann's Organizers in normal HSC and AML by the public database BloodSpot. Intriguingly, the Activin antagonist Follistatin (FST) was significantly increased in hematological malignancies, comparing to the normal HSC. In human malignancies, FST has been shown to express in prostate (Sepporta et al. 2013; Tumminello et al. 2010), ovarian (Ren et al. 2012; Karve et al. 2012; Di Simone et al. 1996), and liver cancers (Patella et al. 2006; Rossmanith et al. 2002) promote cancer cell growth (Gao et al. 2010; Rossmanith et al. 2002). However, the role of FST in human AML and other hematological malignancies is currently unknown. Firstly, we confirmed the upregulation of FST in AML by real-time quantitative PCR, Western Blotting, and immunofluorescence imaging. Mechanistically, FLT3-ITD up-regulated FST via the phosphorylation of CREB as demonstrated by ChIP-PCR. Moreover, the upregulation of FST by FLT3-ITD was effectively blocked by the pharmacological inhibition of CREB in a dosage-dependent manner. FST gene knock-down (by shRNA) or knock-out (by CRISPR/Cas9) in FLT3-ITD+ MOLM-13 AML cell line significantly reduced leukemic growth in vitro, reduced leukemic burden, and prolonged survival of mice in the xenotransplantation experiment. Over-expression of FST in MOLM-13 and ML-2 AML cell lines promoted leukemic growth in vitro and in vivo xenograft mouse model via AKT/p70S6K/mTOR signaling. Furthermore, proteins (RPS19, RPS6, RPS13, RPL6, RPS21, RPSA, RPS18, RPLP2, RPL10A, WIBG, and PPP2R1A) associated with nonsense-mediated decay of mRNA were decreased after FST over-expression in ML-2 AML cells, while the chemoresistant and anti-apoptotic protein CD44 was consistently increased by a label-free proteomics analysis. Notably, the serum FST levels were significantly increased in a patient-derived xenograft AML mouse model. Consistently, plasma FST levels from FLT3-ITD+ AML patients were significantly reduced after an FLT3-ITD inhibitor quizartinib treatment in a phase I/II clinical trial.In conclusion, the CREB targeted gene FST promotes AML cell growth via AKT/p70S6K/mTOR signaling and CD44-mediated anti-apoptosis. Therefore, FST may be a promising therapeutic target for AML treatment. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Translation Initiation and Heat Shock Factor 1 (HSF1): A Novel Strategy Targeting FLT3 Double Mutant and Non-FLT3 Mutant AML Progenitor/Stem Cells

The transcription factor Heat shock factor 1 (HSF1) is a key sensor of proteotoxic stress and plays a major role in cancer biology. In various tumors, downregulation of HSF1 inhibits growth and induces cell death, and its activation was reported to be an adverse prognostic factor in breast and lung cancers. We have reported that inhibition of translation represses DNA-binding of HSF1 in cancers; large scale drug screening identified the eIF4a antagonist rohinitib (RHT) as a potent inhibitor of the HSF1 activation state (Santagata et al, Science 2013). Review of public databases provided us with additional rationale for targeting HSF1 and eIF4a in acute myeloid leukemia (AML): 1) mRNA levels of HSPA8, a primary HSF1 target, are correlated with poor prognosis in AML; 2) eIF4a mRNA levels are highest in AML among 12 cancer types; and 3) Gene set enrichment analysis using microarray dataset of functionally-defined leukemia stem cells (LSCs) and non-LSCs (Nature, Ng et al), based on intra-patient comparisons, reveals that a gene set for translation initiation is highly enriched in LSCs (NES 2.73, FDR = 0.000, p= 0.000).RHT induced marked apoptogenic effects in seven human AML cell lines in culture at low nanomolar concentrations (LD50s; 9.5 to 99.5 nM, IC50s; 4.7 to 8.8 nM, based on AnnexinV/PI-positivity at 72hr). RHT inhibited HSF1 transcriptional activity, as shown by 70% reduction in HSPA8 mRNA levels, and 50% reduction in a luciferase reporter driven by the HSPA1A or HSPA6 promoters. We then tested primary samples from 17 AML patients and bone marrow (BM) samples from eight healthy donors. RHT potently induced apoptosis in AML cells, while relatively sparing normal BM cells (%apoptosis: 65.4% vs 6.67%, P < 0.0001). Importantly, a similarly significant difference in sensitivity was also observed between AML and normal stem/progenitor cell populations (CD45+CD34+CD38-). We further found that the activity of RHT was significantly higher in FLT3-ITD than in FLT3-wt AMLs. Comparing isogenic versions of the cell line Ba/F3, RHT more potently killed FLT3-ITD cells than wild-type (wt) cells (LD50s; 65.3 vs 20.1 nM). Immunoblot analysis showed higher activation-associated phospho-HSF1 (Serine 326) in FLT3-ITD Ba/F3 than in FLT3-wt cells, and growth inhibition by HSF1 knockdown was FLT3-ITD-dependent in those lines, indicating greater dependence of FLT3-ITD cells on HSF1. In an AML xenograft model with GFP-luciferase labeled MOLM-13 cells, RHT significantly reduced tumor burden as measured by bioluminescence imaging and reduction in GFP+ leukemic cells peripheral blood (PB) and BM by day 16. Median survival of the treatment group was significantly longer (24 vs 18 days, p < 0.0001).We also found that RHT was synergistic when combined with FLT3 inhibitors of Type I (sorafenib, quizartinib, midostaurin) or Type II (gilteritinib, and E-6201). Immunoblot analysis showed that oncogenic FLT3 activation is associated with increased phosphorylation of HSF1 (S326), suggesting that decreased phosphorylation of the S326 site associated with FLT3 inhibition and diminished DNA-binding by HSF1 driven by eIF4a inhibition combine to promote synergistic lethality. In support of this, HSF1 knockdown sensitized MOLM-13 and FLT3-ITD+ Ba/F3 cells to FLT3 inhibitors. Importantly, similar combinatorial synergistic effects with type II FLT3 inhibitors were observed in cells with dual mutations of FLT3-ITD and FLT3-D835Y/H. This observation has significant clinical relevance because the development of secondary point mutations is a major resistance mechanism against FLT3 inhibitors.Finally, to test the hypothesis that eIF4a inhibition impairs LSC functions, we treated primary patient-derived xenograft AML PDX cells with RHT ex vivo, then injected identical numbers of live cells into recipient NSG mice. Subsequent leukemia engraftment was delayed by one week vs. controls, and human CD45+ cells in PB and BM at 4 weeks post transplantation were significantly reduced (n = 9, 3.30% vs 0.26% in PB, P < 0.0001). This reduced engraftment capacity of RHT-treated PDX AML-LSCs indicates a toxic effect of eIF4a inhibition on AML-LSCs.Collectively, these preclinical findings strongly support the further development of eIF4a inhibitors for the treatment of AML, especially for high-risk FLT3-mutated AML, with additional potential as LSC-targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

Hypomethylating Agent Leads to Enhanced Immunogenicity of a Dendritic Cell/Acute Myeloid Leukemia Fusion Vaccine and Prolonged Survival in an Immunocompetent Mouse Model

Immunotherapy for acute myeloid leukemia (AML) holds great promise as a modality to overcome tolerance and elicit memory responses needed for durable remissions. Our group pioneered a novel personalized vaccine that targets a broad array of leukemic antigens (Ags) by fusing whole patient-derived AML cells to autologous dendritic cells (DCs). In a clinical trial of AML patients in chemotherapy-induced remission, vaccination induced an expansion of tumor-specific T cells and resulted in prolonged remissions in a subset of patients. Enhancing response to vaccination relies on increasing Ag presentation and overcoming factors in the microenvironment that contribute to tolerance.While the mechanism of action of hypomethylating agents (HMAs) is multifactorial, HMAs are increasingly recognized as exhibiting immunomodulatory functions thought to contribute to therapeutic efficacy. We have previously shown that the HMA SGI-110 augments AML Ag processing and increases AML susceptibility to T cell-mediated killing. In the present study, we aimed to elucidate the effect of HMAs on enhancing Ag presentation and modulating the AML microenvironment as well as to investigate the impact of HMA on the immunogenicity of a DC/AML vaccine.We first investigated the ability of SGI-110 to reverse critical aspects of the AML immunosuppressive milieu and enhance vaccine efficacy. C57BL/6J mice were retro-orbitally inoculated with 1x105 syngeneic TIB-49 AML cells and treated with low-dose SGI-110 (1mg/kg) to assess immunologic effects without preventing engraftment. AML engraftment in the spleen and bone marrow (BM) was similarly observed in treated and control animals. However, SGI-110-treated animals showed decreased PD-1 expression on CD4 T cell splenocytes (mean 3.1% vs. 6.3%; n=4; p=0.01). SGI-110 therapy was also associated with decreased myeloid-derived suppressor cell (MDSC) burden in the BM (n=5; p=0.01) and spleen (n=4; p=0.03) compared to control.Consistent with these findings, treated mice demonstrated increased AML-specific immunity via increased T cells isolated from the BM or spleen that expressed intracellular IFNϒ following ex-vivo exposure to TIB-49 lysate (mean 3% vs. 0.5%; n=5; p=0.02).We then examined the potential synergy of SGI-110 + vaccine in this model. Mice treated with SGI-110 + vaccine showed a significant increase in BM and spleen CD4 (n=5; p=0.05) and CD8 (n=5; p=0.02) T cell IFNϒ in response to tumor lysate as compared to vaccine or SGI-110 alone.To further elucidate the mechanism behind the immune activating effects of HMAs, we investigated the effect of HMA on the endogenous retroviral (ERV) pathway. Activation of this pathway results in increased IFN ß expression leading to increased MHC HLA Class I expression and subsequently, immune stimulation in a solid tumor model (Chiappinelli et al. Cell . 2015). MOLM-14 cells were exposed to low-dose HMA daily X 3 days and analyzed for ERV-3 expression using western blotting (WB). The results showed significant upregulation of ERV-3 expression after treatment. Due to activation of the ERV pathway, increased IFN ß expression was observed with WB analysis. Indeed, analysis of an HLA-A2 positive human AML cell line, THP1, showed significant HLA-A2 upregulation after HMA exposure. Most importantly, HLA-A2 upregulation led to a significant rise in tumor Ag presentation, detected by measuring the binding capacity of a unique T-cell receptor-like antibody using flow cytometric analysis in THP-1 cells.Given these effects, we sought to examine whether treatment with vaccine + SGI-110 would enhance survival in this model. Mice were treated with: vehicle, SGI-110 X 5 days, vaccine, or both agents. Monotherapy with either agent led to a survival advantage compared to untreated mice (p<0.05). Notably, the combination arm further prolonged survival as compared to untreated mice (p<0.05).In conclusion, SGI-110 modulates critical factors contributing to the immunosuppressive milieu in AML by decreasing T cell PD-1 expression and promoting MDSC expansion, resulting in enhanced anti-AML immunity. Moreover, SGI-110 augments the immune response to an autologous DC/AML vaccine in a murine model resulting in a survival benefit. In addition, HMA enhances Ag presentation, in part due to activation of the ERV pathway. The combination of a novel personalized DC/AML fusion vaccine and HMA holds great potential and a clinical trial is being initiated. DisclosuresAvigan:Genus Oncology: Research Funding.

Therapy‐Induced Senescence Is a Predictor of Treatment Outcome in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease, for which biologically grounded predictors of outcome remain an urgent clinical need. In addition to tumor cell death, antineoplastic drugs have been shown to mediate a long-lasting growth arrest of vital, metabolically active tumor cells termed therapy-induced senescence (TIS) in various cancer entities and model systems, but predictive investigations on TIS are lacking. We hypothesized here that an ex vivo assessment of TIS in blast samples from AML patients at diagnosis might serve as a biomarker of outcome.Methods: We established assays to quantitatively detect TIS in a primary culture setting of purified primary human AML specimens (n > 30) assessed through cytochemical and fluorescence-based analysis of senescence-associated β-galactosidase (SA-β-gal) activity and other senescence markers (such as p16INK4a, H3K9me3) in concert with downregulation of proliferation markers (Ki67, EdU). AML samples were subjected to standard karyotype and molecular genetics analyses, defining risk groups according to the European Leukemia Net (ELN) genetic risk stratification.Results: We found the individual fractions of senescent AML blasts in response to the standard anti-leukemic agents Daunorubicin and Cytarabine samples to be positively correlated with better disease-free (p= 0.042) and overall survival (p= 0.004) of the patients. Furthermore, a more favorable molecular risk group (p= 0.005), normal karyotype (p= 0.007), and NPM1 mutations (p= 0.018) as well as DNMT3A mutations (p < 0.001) were associated with higher TIS levels in our ex vivo senescence assay.Discussion: Our study is the first report providing direct evidence that cellular senescence induced in patient-derived AML blasts by chemotherapeutic drugs ex vivo serves as a predictor of patient long-term responsiveness to standard induction therapy. TIS might explain the underlying biology of current para-clinical risk indicators, and also seems to identify subgroups of superior outcome in pre-defined risk groups including patients with DNMT3A-mutant disease. Additional investigations exploring senescence in response to other AML-relevant agents and functional analyses expanding to patient-derived xenograft (PDX) AML models will be presented at the meeting. DisclosuresNo relevant conflicts of interest to declare.

Blocking CD70/CD27 Signaling in Combination with Hypomethylating Agents Eradicates Human CD34+ AML Stem and Progenitor Cells in Vitro and In Vivo

Leukemia stem cells (LSCs) are the origin of acute myeloid leukemia (AML) and are resistant to standard therapeutic regimens resulting in frequent relapse of the disease and poor prognosis especially for AML patients not fit for intensive chemotherapy. Consequently, LSCs represent a major obstacle for AML therapy. We recently identified the interaction of the TNF ligand CD70 and its receptor CD27 on LSCs as a promising therapeutic strategy to target LSCs.In this study, we demonstrate for the first time that treatment with hypomethylating agents (HMA, azacitidine or decitabine) significantly up-regulates CD70 mRNA and protein expression on primary CD34+ stem/progenitor cells from newly diagnosed AML patients independent of the cytogenetic/molecular risk category in vitro (mRNA: 4.3±1.6 fold increase, FACS: 4.0±0.9 fold increase vs. vehicle treatment, n=15). Similarly, CD34+ stem/progenitor cells from AML patients after 5 days decitabine administration (20mg/kg by continuous intravenous infusion over 1 hour repeated daily) exhibited a significant increase in CD70 expression as analyzed by flow-cytometry (6.8±1.1 fold increase vs. diagnostic sample, n=6). In contrast, CD70 expression of CD34+ stem/progenitor cells from healthy donor bone marrow (n=3) was not affected by the treatment.Consequently, co-treatment of CD34+ stem/progenitor cells from AML patients of each risk group (n=3) with the HMA decitabine and a blocking αCD70 monoclonal antibody strongly reduced colony-forming and re-plating capacity in vitro compared to single agent treatment. To confirm and extend our findings towards disease-initiating CD34+ AML LSCs in vivo, we performed patient-derived AML xenografts (PDX) from one patient with intermediate and one patient with adverse cytogenetic risk profile. Combining decitabine treatment with CD70 blockade significantly reduced AML engraftment in blood, spleen and BM compared to controls. Moreover, co-treatment significantly reduced frequency and absolute numbers of CD34+CD38- and CD34+CD38-CD45RA- AML LSCs in the BM as analyzed phenotypically by FACS and eliminated human LSCs as analyzed in limiting dilution PDX experiments compared to single treatments. Importantly, CD34+ hematopoietic stem/progenitor cells from healthy human bone marrow (n=3) were only marginally affected by the co-treatment.In summary, combining HMAs with blocking CD70/CD27 signaling could represent a novel strategy to eradicate human LSCs. DisclosuresVan Rompaey:argenX: Employment. Leupin:argenX: Employment. De Haard:argenX: Employment.