Leukemic Stem Research Articles

Spatially Resolved Transcriptomics Unveils Unique T-Cell Dysfunctional States and Prognostic Gene Expression Signatures in TP53-Mutated Acute Myeloid Leukemia

Introduction Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 8% to 10% of AML and correlate with primary induction failure, high risk of relapse, and dismal prognosis. We have previously shown that TP53 abnormalities are associated with a pro-inflammatory and immunosuppressive tumor microenvironment (TME), including high CD274 and FoxP3 expression, with dysfunctional natural killer (NK)/CD8 + T-cell states and with response to flotetuzumab, a CD123 × CD3 bispecific T-cell engager. In the current study, we explore the cellular neighborhoods of TP53-mutated ( TP53-m) AML using spatially resolved transcriptomics and we employ machine learning to identify prognostic RNA signatures. Methods We analyzed the expression of >1,800 immune and TME genes in bone marrow (BM) formalin-fixed paraffin-embedded (FFPE) sections (5-µm) from a discovery cohort of 11 patients with chemotherapy-naïve AML (N=5 TP53-m and N=6 TP53-wt cases) using the GeoMx™ Digital Spatial Profiling (DSP) platform (Cancer Transcriptome Atlas with next generation sequencing read-out; NanoString Technologies, Seattle, WA). Imaging was performed at 20× magnification and a total of 151 circular regions of interest (ROIs) with a 300-600 µm diameter were selected (Fig. 1A). We then generated a ROI-by-gene count matrix for deconvolution and downstream pathway analysis. For each ROI, enrichment scores reflecting immune senescence/dysfunction and leukemia stemness were calculated with gene set variation analysis (GSVA), using a manually curated compendium of published gene signatures as input. We also employed public bulk and single-cell RNA-sequencing (sc-RNA-seq) AML cohorts for in silico modeling and validation. Results We initially captured transcripts from ROIs of TP53-m and TP53-wt AML with varying degrees of BM infiltration with T cells (CD3 hotspots) and AML blasts, and we found that TP53-m is enriched in T cell-specific, type I/II interferon (IFN) and pro-inflammatory genes ( IL1B), as well as leukemia stem cell (LSC)-related gene programs (LSC17). After dichotomizing ROIs based on a median split of CD3D RNA expression, we observed that T-cell low ROIs express higher levels of monocyte-macrophage and myeloid derived suppressor cell (MDSC)-related genes, including S100A9 and S100A8. These findings point to previously unappreciated links between BM infiltration with immune-modulatory myeloid cell types and leukemia stemness / T-cell abundance. We then tested for differential cell density in neighborhoods (regions of transcriptionally similar cells) across TP53-m and TP53-wt AML using MiloR in a sc-RNA-seq AML dataset annotated by cell type (N=42 cases, of which N=3 with TP53-m; Lasry A, et al. Nat. Cancer 2023). This orthogonal approach highlighted a significantly higher frequency (FDR <0.05) of multipotent and granulocyte-macrophage progenitor neighborhoods in TP53-m AML, alongside suppression of adaptive immune system pathways (Fig. 1B). We next inferred active receptor-ligand pairs mediating the crosstalk between T cells and leukemic blasts in the TME of TP53-m AML, and we uncovered decreased MHC class I/II-mediated signaling, curbed CD8 +/CD4 + T cell-to-AML interactions and enhanced CD6/ CDCP1( CD318) signaling (Fig. 1C). We also found that CD8 + and CD4 + T cells from TP53-m AML are enriched in effector memory (T EM) / terminally differentiated effector memory (T EMRA) and regulatory T-cell gene programs, respectively, compared with their cellular counterparts from TP53-wt patients (Fig. 1D). In particular, T EM cells from TP53-m AML expressed genes known to regulate T-cell dysfunctionality, foremost PRDM1, and transcription factors ( ZEB2) expected to drive the differentiation of killer cell lectin-like receptor (KLR) + exhausted T-cell states with residual cytolytic potential. Finally, we applied the least absolute shrinkage and selection operator (LASSO) statistical method for feature selection to our discovery AML dataset, and we constructed a TP53-m-related ‘spatial metagene’ that predicted poor overall survival in TCGA-LAML (N=170) and Beat-AML2 cases (N=444; Fig. 1E). Conclusions Spatial gene programs of leukemia stemness, T-cell dysfunction and immune suppression are enriched in TP53-m AML. It remains to be determined whether TP53-m AML with a pre-existing but ineffective T-cell response may be amenable to T cell-targeting immunotherapies.

UBE2N Regulates Oncoprotein Networks in Myeloid Malignancies

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) originate from clonal mutant hematopoietic stem and progenitor cells, referred to as leukemic stem and progenitor cells (LSPCs). Ubiquitination is an essential process for various cellular maintenance functions, including protein degradation and scaffolding, which is often perturbed in a variety of cancers. Ubiquitination of protein substrates involves three related enzymes: the E1 activating enzyme, the E2 conjugating enzyme, and the E3 ligase. In humans, there are up to 40 E2 conjugating enzymes, among which Ubiquitin conjugating enzyme E2 N (UBE2N) stands out as one of the most crucial and overexpressed E2 enzymes in AML cells. Our recent study reported that UBE2N is a druggable target in MDS and AML (Barreyro et al., Science Translational Medicine, 2022), and we found that its catalytic active site cysteine 87 is required for various AML mouse models, including MLL-AF9, FLT3-ITD, AML1-ETO9a, and MN1 (unpublished). These observations indicate that the catalytic function of UBE2N is critical for AML cells but dispensable for normal hematopoiesis. To examine the active state of UBE2N, we investigated the “charged” version of UBE2N, which contains a ubiquitin bound to cysteine 87, in normal hematopoietic cells and in a AML cell line panels. Our findings confirmed that the activated form of UBE2N is highly expressed in human AML cells compared to normal hematopoietic cells. UBE2N exclusively synthesizes Lysine-63 (K63)-linked polyubiquitin chains on target proteins in conjunction with selected E3 ligases, leading to the stabilization and/or activation of protein substrates. To identify the relevant targets of UBE2N in AML, we conducted a ubiquitin-enrichment followed by a proteomic analysis in isogenic UBE2N-deficient AML cells compared to WT AML cells. We identified ~300 proteins that are ubiquitinated by UBE2N. Importantly, UBE2N regulates a network of leukemia-associated oncoproteins, including SYNCRIP, HUWE1, NPM1, TYK2, IKBKG, VAV1, BAX, IGF2BP2, IRF4, IRAK4, RBMX, STAT3, and BTK. UBE2N can interact with various E3 ligases in a cell- and context-dependent manner. To gain insight into the major E3 ligase that cooperates with activated UBE2N in AML, we performed a proximity-based labeling assay followed by mass spectrometry. This interactome screen identified Tripartite motif containing 21 (TRIM21) as the top E3 ligase that partners with UBE2N in AML cells. TRIM21 is overexpressed in AML patients compared to healthy controls. Moreover, deletion of TRIM21 in AML cells impaired cell viability and proliferation in vitro and prolonged survival in a xenograft model in vivo, which phenocopies the loss of UBE2N. Importantly, restoration of TRIM21 rescued the functional defect of UBE2N-deficient AML cells, indicating that the UBE2N-TRIM21 axis is operational in AML. To prioritize the UBE2N-dependent oncoprotein network, we focused on UBE2N-TRIM21 regulated signaling in AML. For this, a gene expression analysis was performed in UBE2N-deficient AML cells that were restored with TRIM21. The top UBE2N-TRIM21 oncoprotein network that emerged was related to interferon-STAT signaling. Additionally, both UBE2N and TRIM21 were found to regulate STAT3 ubiquitination. Deletion or inhibition of UBE2N catalytic activity significantly reduced total and activated STAT3 in AML cells. Finally, the expression of constitutively active STAT3 partially restored the functional defect of AML cells treated with a UBE2N inhibitor, indicating that STAT3 function, a known driver of LSPCs, is regulated via the UBE2N-TRIM21 axis in AML. In summary, our data reveal that UBE2N regulates oncoprotein networks in AML and that inhibiting UBE2N catalytic function, such as with small molecule inhibitors, effectively suppresses LSPCs.

Synergy of Stag2-Cohesin Loss Results in Expansion of Npm1c-Mutant Hematopoietic Stem and Progenitor Cells

STAG2 is a member of cohesin complex that is recurrently mutated in >10 cancers and is essential in maintaining the integrity of the 3D genome partitioning structure known as topologically structural domains (TADs). Our previous work has demonstrated that depletion of various cohesin factors, including Stag2, leads to increased hematopoietic stem and progenitor population (HSPC) self-renewal and myeloid-biased differentiation. Loss of Stag2 leads to impaired sub-TADs and affects key hematopoietic transcription factors, such as PU.1, to access and engage their target genes. Yet, the extent and cooperative transformational role of Stag2-cohesin with leukemia driver mutations, such as Npm1c, remains unexplored. To determine whether Stag2-cohesin regulates leukemic 3D chromatin organization, we generated dual Stag2 Δ Npm1 c/+ murine models with tamoxifen inducible UbcCreER T2. After 4 weeks, HSPCs (LSK cells; Lin-Sca1+Kit+) are increased in Stag2 Δ Npm1 c/+ double mutant mice ( Figure 1A). Within LSK, Stag2 Δ Npm1 c/+ but not Npm1 c/+ has marked expansion of the myeloid bias MPP2 (LSK+Flk2-Cd150+Cd48+) and MPP3 (LSK+Flk2-Cd150-Cd48+) compartment. Interestingly, Stag2 Δ single mutant exhibit LSK and MPP3 expansion at 16 weeks, suggesting the early expansion of HSPC compartment is the result of Stag2 ΔNpm1 c/+ synergistic acceleration. No difference in serial replating of LSK and GMP (LSK-Cd34+FcyRII+) cells were observed between Npm1 c/+ and Stag2 Δ Npm1 c/+. However, Stag2 Δ Npm1 c/+ LSK cells had impaired reconstitution capacity and myeloid biased output in primary and secondary transplantation. To determine if the blockage is specific to Stag2-cohesin loss, we generated the Smc3/Npm1c double mutant mice, where both Stag1-cohesin and Stag2-cohesin complexes were partially depleted upon heterozygous deletion of Smc3. Interestingly, there were no changes in LSK or MPP3 population in Smc3 +/- Npm1 c/+ mice, suggesting it is specific to Stag2-cohesin loss which obstructs differentiation in Npm1 c/+ HSPC. Mechanistically, we performed bulk ATAC-seq on sorted HSC LT (LSK+Flk2-Cd150+Cd48-) and MPP3 population, as poor long-term reconstitution suggests functional alteration in HSC LT. Comparing to WT, both Stag2 Δ and Stag2 Δ Npm1 c/+ HSC LT have increased chromatin accessibility, while only Stag2 Δ Npm1 c/+ MPP3 have persistent opening of chromatin. Motif analysis of increased accessible peaks identified RUNX2, GATA3 and MEIS1 are among the increased accessible region in Stag2 Δ Npm1 c/+ MPP3, which reflects the increased myeloid output in the transplantation. Recently, various group have suggested that Npm1c directly binds to chromatin and drives the overexpression of genes, especially at HOX clusters and MEIS1. This suggests a possible synergistic mechanism which Stag2-cohesin regulates chromatin accessibility of key TF loci, which influences normal and leukemic stem and progenitor differentiation. To comprehensively profile the LSK population, we performed scRNAseq at 4 weeks post mutation activation, results showed a marked immune pathway activation in Stag2 Δ Npm1 c/+ LSK cells. To inter cell fate transition, we employed CellDancer, an RNA velocity based algorithm and found that Stag2 Δ Npm1 c/+ MPP cells have altered differentiation trajectory and delayed kinetics at MPP3 stages, suggesting a differentiation block ( Figure 1B). In conjunction with our results, we postulate that Stag2-cohesin regulates TF interactions with regulatory elements during cell fate transitions. We are currently investigating the direct interaction of Stag2-cohesin with regulatory elements in Npm1 c/+ cells and compare to WT and Stag2 Δ Npm1 c/+ cells. We will investigate the 3D genome organization of Npm1 c/+ and Stag2 Δ Npm1 c/+ cells to determine whether Stag2 loss induces the phenotypic changes via Ctcf-dependent or independent manner.

Safety, Pharmacodynamic, and Anti-Tumor Activity of SL-172154 As Monotherapy and in Combination with Azacitidine (AZA) in Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) and Higher-Risk Myelodysplastic Syndromes/Neoplasms (HR-MDS) Patients (pts)

Background: CD47 blocking antibodies and SIRPα-Fc fusion proteins have demonstrated activity in combination with azacitidine (AZA) in previously untreated (UnTx) HR-MDS and AML pts. SL-172154 (SIRPα-Fc-CD40L), a hexameric, bi-functional fusion protein consisting of SIRPα domains linked to CD40L domains through an inert Fc linker demonstrated improved anti-tumor activity in comparison to naked CD47 blocking antibodies in pre-clinical studies [de Silva et al. Cancer Immunol Res 2020]. SL-172154, as a CD47 inhibitor, requires combination with AZA to enhance pro-phagocytic signals on leukemic stem cells/blasts thereby potentiating macrophage phagocytosis in AML/HR-MDS. Here we report the results from the Phase 1 Dose Escalation. cohorts for SL-172154 monotherapy [SL-mono] and for SL-172154 + AZA combination [SL-AZA] in pts with HR-MDS or AML. Methods: The objectives of the dose escalation cohorts include evaluation of safety, dose-limiting toxicity (DLT), recommended phase 2 dose, pharmacokinetic and pharmacodynamic effects and efficacy per IWG 2006 for MDS and ELN 2017 for AML. SL-172154 was administered IV weekly of a 28-day cycle as monotherapy or with AZA 75 mg/m 2 for 7 days per cycle. mTPI-2 was used for dose escalation with a DLT threshold of 20%. Pts ≥ 18 years old with R/R HR-MDS or R/R AML were eligible for dose escalation cohorts. In addition, UnTx pts with TP53 mutant (TP53m)-MDS were eligible for SL-AZA cohorts. Results: As of May 25, 2023, 37 pts were enrolled (19 in SL-mono; 18 in SL-AZA cohorts): 14 women; median age 70 years [range 44-81]. 27 pts had R/R AML (16 de novo; 11 secondary), 5 had R/R MDS, and 5 were UnTx TP53m-MDS. 23 (85%) of 27 R/R AML pts had adverse genetic abnormalities per 2017 ELN classification, including 9 (33%) with TP53m or del 17. The median number of prior lines of AML therapy was 2 [range 1-4]; 25 (93%) had received venetoclax (VEN) and 23 (85%) had received a hypomethylating agent (HMA). All 5 pts with R/R MDS had previously received HMA and 2 pts were previously treated with VEN. SL-172154 was dose escalated as monotherapy or with AZA as shown in Table 1. Infusion-related reactions (IRRs) were the most common SL-172154-related treatment-emergent AEs (TEAEs) reported in 13 (68%) and 8 (44%) pts in SL-mono and SL-AZA cohorts, respectively. IRRs (51 events) were Grade (G) 1 or 2 except for 3 G3 events (1 each at 6 mg/kg SL-mono and SL-AZA; 1 at 3 mg/kg SL-mono). Other SL-172154-related TEAEs observed in ≥ 3 pts were AST increased (4; 21%), ALT increased (3; 16%) and nausea (3; 17%) in SL-mono cohorts, and nausea (3; 17%) in SL-AZA cohorts. All events of AST/ALT increased were transient. Only one DLT (G3 IRR in the 6 mg/kg SL-AZA cohort) was reported, which required a dose reduction to 3 mg/kg. As of July 10, 2023, in SL-mono cohorts, 1 pt with R/R AML post 7+3, FLAG and VEN/AZA achieved Morphologic Leukemia-Free State (blast reduction from 19% to <5%) after 1 cycle of 6 mg/kg SL-172154 and proceeded to allogeneic hematopoietic cell transplant (allo-HCT) after cycle 2. In 12 evaluable pts with R/R AML receiving SL-AZA, although no objective responses (OR) were observed, relative reduction in bone marrow (BM) blasts from baseline was reported in 2/5 pts at 1 mg SL-AZA (-50%, -75%) and 5/7 pts at 3 mg SL-AZA (ranging from -35% to -90%). One pt underwent an allo-HCT. In 4 evaluable pts with UnTx TP53m-MDS, the ORs were 1 complete remission (CR), 1 marrow CR, and 2 stable disease. 2 of them proceeded to allo-HCT. SL-172154 induced elevations in serum IL-12p40, IP-10, IL-8, IL-10, MIP3α and MCP1: greater response at 3 mg/kg compared to 1 mg/kg while similar between 3 and 6 mg/kg. In the BM, SL-172154 induced a dose-dependent increase in phagocytic cells (CD11b, HLA-DR, CD16, CD36 and CD64) within mature myeloid immune cell compartments (CD45highCD34-). Reduction in leukemic blasts (CD45lowCD34+) was associated with an increase in mature myeloid and phagocytic cell phenotypes (3 mg/kg SL-AZA > 1 mg/kg SL-AZA). Conclusions: SL-172154 was well tolerated up to 3 mg/kg as monotherapy and in combination with AZA. Preliminary efficacy signals were detected in UnTx TP53m-MDS and R/R AML. A response to SL-172154 monotherapy, dose-dependent increases in serum cytokines and accumulation of mature myeloid cells in BM suggest a potential role for CD40 stimulation. Based on the safety, preliminary efficacy and pharmacodynamic activity, 3 mg/kg SL-172154+ AZA is being evaluated in treatment naïve pts with TP53m AML and HR-MDS.

Monoclonal Antibodies Targeting LIGHT Block Ltbr Signalling and Eliminate Acute Myeloid Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is a molecular heterogeneous disease that originates from the malignant transformation of a hematogenic stem- or progenitor cells resulting in a leukemia stem cell (LSC). LSCs are a main cause of resistance to therapy and relapse. Despite recent advances in targeted therapy and immunotherapy, prognosis for elderly patients not fit do undergo intensive therapy remains poor. We recently documented that targeting CD70, the ligand of the tumor necrosis factor receptor (TNFR) CD27, by the antibody-dependent cell-mediated cytotoxicity (ADCC)-enhanced monoclonal antibody (mAb) cusatuzumab eliminates LSC. We now studied the role of the related TNFR lymphotoxin b receptor (LTbR) with its ligand LIGHT in the pathogenesis of AML in mice and humans and developed a LIGHT targeting mAb to specifically eliminate LSCs. Experimental design We investigated the impact of LIGHT/LTbR signalling in AML development by using the retrovirally-induced MLL-AF9 syngeneic AML mouse model. Light-, Ltbr -deficient and control MLL-AF9 transduced LSCs were injected intravenously into non-irradiated BL/6 recipient mice. AML development was evaluated by analysing MLL-AF9-GFP-positive leukemic cells in blood, spleen, and bone marrow (BM). BM LSC were phenotypically characterized by FACS and functionally by performing colony forming unit (CFU) assays in vitro and by secondary transplantations of LSCs including limited dilution assays (LDA) in vivo. Correlative survival analysis for LIGHT and LTbR expression was performed on a publicly available gene dataset (GSE6891). LIGHT and LTBR protein expression was analysed on primary AML CD34 + leukemic stem and progenitor cells (LSPCs) and control human CD34 + hematopoietic stem cell and progenitor cells (HSCPs). LIGHT/LTbR signalling was blocked by either siRNA treatment or by treatment with novel LIGHT-targeting mAb. The effect of blocking LIGHT/LTbR-signalling was analysed in vitro by CFU assays, RNA-sequencing and xenotransplant experiments in vivo. LIGHT-targeting mAbs were generated by immunization of llamas and phage display selections followed by cloning of FABs into a human backbone (hIgG1). Clones were selected based on their blocking potency of receptor/ligand interaction using competition ELISA and FACS-based cell binding affinity assays. ADCC-enhanced anti-LIGHT mAbs were generated via POTELLIGENT® technology. Anti-LIGHT mAbs that specifically block the interaction of LIGHT/LTBR with deficiency in effector functions (Fc-death) were produced by amino acid substitutions in the CH2 domain of the parental clone. Results LTbR was expressed by HSCs and AML LSCs. In contrast, LIGHT was only expressed on murine and human LSCs but not normal HSCs. The absence of LIGHT or its receptor LTbR on LSCs resulted in significantly prolonged survival and decreased numbers of LSC in BM of AML mice. Light -/- and Ltbr -/- LSCs had a reduced CFU capacity compared to control LSCs. Similarly, inhibition of LIGHT-signalling on LSCs via LTbR-Ig significantly reduced colony formation. Moreover, secondary transplantation of Light -/-LSCs resulted in prolonged survival compared to Light-proficient LSCs. An LDA analysis revealed a 11-fold reduction of the LSC frequency in BM in the absence of LIGHT or LTBR. Similarly, inhibiting LIGHT signalling by siLIGHT RNA in human AML LSPCs resulted in reduced CFU capacity compared to control siRNA treatment. In addition, a GEO analysis revealed that low LIGHT or LTbR mRNA levels correlated with improved overall survival in AML patients. After immunizations of llamas and phage-display selection three humanized anti-LIGHT mAbs with high blocking and high binding efficiency were selected (mAb10F11 and 12C2 specific for human LIGHT and the cross-reactive murine/human 9F6 mAB). Incubation of LSCs with the blocking mAbs significantly reduced CFU capacity. HSPCs from healthy donors remained unaffected by the treatment. A gene expression analysis revealed that blocking LIGHT/LTbR-signalling in LSCs reduced stemness and promoted differentiation. In addition, ADCC-enhanced mAbs activated NK cells to eliminate LIGHT-expressing LSCs. Our findings reveal that LIGHT/LTbR-signalling is crucial for the pathogenesis of AML and especially for the maintenance and expansion of LSCs. We developed and validated novel LIGHT targeting mAbs in vitro and in vivo for further development in clinical trials.

Glucose partitioning in the bone marrow micro-environment in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) cells metabolise glucose by glycolysis-based re-programming. However, how glucose uptake is partitioned between leukaemia cells and other cells of the bone marrow micro-environment is unstudied. We used a positron emission tomography (PET) tracer 18F fluorodeoxyglucose ([18F]-FDG) probe and transcriptomic analyses to detect glucose uptake by diverse cells in the bone marrow micro-environment in a MLL-AF9-induced mouse model. Leukaemia cells had the greatest glucose uptake with leukaemia stem and progenitor cells having the greatest glucose uptake. We also show the effects of anti-leukaemia drugs on leukaemia cell numbers and glucose uptake. Our data suggest targeting glucose uptake as a potential therapy strategy in AML if our observations are validated in humans with AML.

Triple combination targeting methyltransferase, BCL-2, and PD-1 facilitates antileukemia responses in acute myeloid leukemia.

A recent breakthrough therapy combining the BCL-2 inhibitor venetoclax with hypomethylating agents (HMAs) targeting DNA methyltransferase has improved outcomes for patients with acute myeloid leukemia (AML), but the responses and long-term survival in older/unfit patients and in patients with relapsed/refractory AML remain suboptimal. Recent studies showed that inhibition of BCL-2 or DNA methyltransferase modulates AML T-cell immunity. By using flow cytometry and time-of-flight mass cytometry, the authors examined the effects of the HMA decitabine combined with the BCL-2 inhibitor venetoclax (DAC/VEN therapy) on leukemia cells and T cells in patients with AML who received DAC/VEN therapy in a clinical trial. The authors investigated the response of programmed cell death protein 1 (PD-1) inhibition in the DAC/VEN-treated samples in vitro and investigated the triple combination of PD-1 inhibition with HMA/venetoclax in the trial patients who had AML. DAC/VEN therapy effectively targeted leukemia cells and upregulated the expression of the immune checkpoint-inhibitory receptor PD-1 in T cells while preserving CD4-positive and CD8-positive memory T cells in a subset of patients with AML who were tested. In vitro PD-1 inhibition potentiated the antileukemia response in DAC/VEN-treated AML samples. The combined use of azacitidine, venetoclax, and nivolumab eliminated circulating blasts and leukemia stem cells/progenitor cells and expanded the percentage of CD8-positive memory T cells in an illustrative patient with relapsed AML who responded to the regimen in an ongoing clinical trial. Immunomodulation by targeting PD-1 enhances the therapeutic effect of combining an HMA and venetoclax in patients with AML.

The Novel Bispecific Innate Cell Engager (ICE®) AFM28 Efficiently Directs Allogeneic NK Cells to CD123+ Leukemic Stem- and Progenitor Cells in AML

Introduction: Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by expansion of undifferentiated myeloid cells in the bone marrow (BM) and peripheral blood. Although AML can be treated with curative intent, residual leukemic stem cells (LSCs) are frequently reservoirs for relapse, which is associated with a poor prognosis. Moreover, especially elderly patients are often not eligible for intensive treatment regimen. Therefore, there is a high need for novel treatment strategies, which are capable of eliminating LSCs in order to induce long-term remissions, suppress measurable residual disease (MRD) and prevent relapse. A promising emerging approach is the adoptive transfer of allogeneic natural killer (NK) cells, which was shown to be clinically active and could induce complete remissions in relapsed/refractory (R/R) AML. Innate Cell Engagers (ICE®) may further augment the activity of NK cell therapies, in particular against LSCs and hold the potential to increase frequency and durability of responses. The tetravalent bispecific CD123/CD16A ICE®, AFM28, directs the cytotoxic activity of CD16A-expressing NK cells towards CD123-expressing (CD123+) AML blasts and LSCs. In this study, we evaluated the efficacy of AFM28 in pre-clinical models of AML. Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) data were obtained by a 4h calcein release assay using AML cell lines and primary human NK cells (E:T ratio 2.5:1) in the presence of titrated antibodies. CD64 and CD123 expression levels on AML cell lines were evaluated by flow cytometry. Intracellular FACS was used to detect STAT5/pSTAT5. Proliferation of TF-1 cells in the presence of IL-3 or GM-CSF was measured via CellTiter Glo Assay (Promega). Using primary BM samples of n=6 AML patients and n=4 healthy donors, ADCC assays and colony-forming unit (CFU) assays were performed. BM-MNC (ADCC assay) or enriched CD34+ cells (CFU assay) and allogeneic NK cells (E:T ratio 1:1) were treated with AFM28 or an Fc-enhanced anti-CD123 antibody comparator at different concentrations of AFM28 (0-500 or 0/10/100/1000 pM, respectively) for 24h. Subsequently, ADCC assays were analyzed by flow cytometry and CFU assays were evaluated after 7-14 days. Systemic anti-tumor activity of AFM28 in vivo was assessed using in-life imaging (BLI) in a T cell-depleted huFcgR-C57BL6 transgenic mouse model against intravenously administered luciferase-transfected murine AML cells expressing human CD123 as tumor antigen. Results: Pre-clinical in vitro models using a panel of AML cell lines showed efficacious ADCC against CD123+ tumor cells by allogeneic NK cells in the presence of AFM28. ADCC induction by AFM28 was independent of leukemic cell mutational profiles and even targeted cells with low levels of CD123 surface expression. Of note, expression of the high-affinity IgG receptor CD64 on AML cell lines completely abolished ADCC induction by an Fc-enhanced anti-CD123 antibody whereas AFM28 also showed high ADCC efficacy against all tested CD64+ cell lines. In addition, AFM28 exerted an NK cell-independent inhibitory effect on the IL-3-induced phosphorylation of STAT5 and abolished proliferation of CD123+ TF-1 cells, indicating an antagonistic effect on IL-3R signaling. In ex vivo models using primary AML patient cells, AFM28 demonstrated strong ADCC mediated by allogeneic NK cells. CFU assays of patient-derived AML BM samples performed subsequent to incubation with allogeneic NK cells with and without AFM28 showed significantly reduced numbers of outgrowing colonies in AFM28 treated samples, indicating that AML LSCs and progenitor cells were eliminated. In the in vivo efficacy study with huFcgR-C57BL6 mice, treatment with AFM28 at 15 mg/kg b.w. led to complete inhibition of tumor growth in 5/5 animals throughout a treatment period of 42 days. In contrast, all untreated control mice (6/6) developed systemic disease. Conclusion: AFM28 induced highly potent and selective lysis of CD123+ leukemic cells, including LSCs and progenitor cells, by allogeneic NK cells in vitro and in vivo. The capacity to eradicate LSCs holds the promise for durable responses and the potential for long-term remissions in patients with R/R AML. AFM28 is currently being prepared for a first-in-human clinical investigation as monotherapy and in combination with allogeneic NK cells.

CD69 Marks Leukemic Regenerating Cells and Regulates Their Metabolism in AML

The treatment of relapsed or refractory (R/R) acute myeloid leukemia (AML) has been a challenge for several decades. Despite the widespread use of cytarabine-based chemotherapy and novel targeted agents, the 5-year overall survival (OS) of AML is still only about 30%, and patients mostly die from relapsed AML. Single-cell RNA sequencing (scRNA-seq) has been applied in the study of recurrence and metastasis of solid tumors, but there is still a lack of in-depth exploration of AML. Here, we investigated the transcriptome features of leukemia stem cells (LSCs) in R/R AML, explored their markers and mechanisms of relapse and chemoresistance, and provided potential therapeutic targets for the clinic. To explore the features of LSCs in R/R AML, we collected 13 bone marrow samples for scRNA-seq, including 8 samples from 4 AML patients at the time of initial diagnosis and their relapse after chemotherapy between 2017 and 2021 (median time to relapse was 333.5 days), 2 samples at initial diagnosis from AML patients with relapse-free survival for three years after chemotherapy, and 3 healthy donors (Figure.1A). Through clustering algorithm, activity analysis of relugons and copy number variation analysis comparing with normal bone marrow samples, we isolated AML cells from normal cells (Figure.1B). Changes of cell number of each cluster and leukemia stemness gene scores were used to define stemness-deficient leukemia cells (Bulk), chemotherapy-sensitive LSCs (S-LSC), and resistant LSCs (R-LSC) (Figure.1C-D). Compared with S-LSCs or Bulk, R-LSCs significantly upregulated the oxidative phosphorylation pathway (Figure.1E). Besides, we found that CD69 was significantly upgraded in R-LSCs, indicating that CD69 may represent a group of leukemic-regenerating cells (LRCs) associated with both leukemic stemness and chemoresistance (Figure.1F) . To investigate the impact of upregulated genes in R-LSC on the clinical prognosis of AML, we explored GEO database and found that the highly expressed genes in R-LSCs including CD69 were associated with a worse clinical prognosis (Figure.1G). TCGA database was used as a validation, and consistent results were obtained (Figure.1H). To explore the stemness, chemoresistance, and metabolic function of CD69+ LRCs, we sorted CD69- and CD69+ AML cells from patients. Through in vitro chemotherapy, we found that CD69+ AML cells were more resistant to chemotherapy than CD69- AML cells (Figure.2A). By clone formation assay, we found that CD69+ AML had better clonal proliferation ability (Figure.2B). To explore the stemness of CD69+ AML cells, we performed limiting dilution analysis (LDA) in vivo and found that the LSCs ratio of CD69+ AML was 109 times that of CD69- AML (p < 0.0001) (Figure.2C). Next, we explored the metabolic function of CD69+ AML cells and found that CD69+ AML cells had higher levels of pyruvate and ATP metabolism, while lactate metabolism levels were lower than those of CD69- AML cells (Figure.2D-F). After culturing with the ATP synthase inhibitor, the chemosensitivity of CD69+ AML cells was significantly improved (Figure.2G). The above evidence proves that CD69 marks a population of LRCs with high oxidative phosphorylation, stemness and chemoresistance in AML, which is consistent with the scRNA-seq results. Based on the above conclusions, we further explored the mechanism by which CD69+ LRCs maintain chemoresistance properties. By scRNA-seq analysis, we found that CD69+ LRCs had enhanced activity of STAT5 in the regulatory network (Figure.3A). We found the expression of phospho-STAT5A, MYC and BCL-2 proteins in CD69+ AML cells of patients was significantly higher than that in CD69- AML cells. To explore whether the function of CD69+ LRCs is affected by the JAK3-STAT5 pathway, we knocked down STAT5 in human AML cells. The protein expressions of MYC and BCL-2 were significantly reduced (Figure.3B). The chemosensitivity in vitro was significantly reduced in STAT5 knockdown (KD) (Figure.3C). To explore the effect of STAT5 KD on the metabolism of CD69+ LRCs, we detected metabolic index. We found that CD69+ LRCs with STAT5 KD produced decreased levels of pyruvate (Figure.3D). In conclusion, CD69 markers LRCs with high oxidative phosphorylation. CD69+ LRCs affect their metabolism and stemness through JAK3-STAT5 activation of downstream MYC and BCL-2. CD69-targeted and STAT5-targeted inhibitors may be candidate strategies for clinical R/R AML treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase 1b Study of IDH Inhibition with Enasidenib and MEK Inhibition with Cobimetinib in Patients with Relapsed or Refractory Acute Myeloid Leukemia Who Have Co-Occurring IDH2 and RAS Signaling Gene Mutations

Background: For patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) with IDH2 and RAS-pathway mutations involving NRAS, KRAS, PTPN11, and CBL genes, low responses and poor survival remain unmet clinical needs. Although enasidenib, a potent inhibitor of IDH2, is approved in R/R IDH2-mutant AML, patients with co-occurring RAS-pathway mutations exhibit primary resistance.[1,2] , Additionally, new RAS mutations confer acquired resistance to IDH inhibitors.[3] Preclinical studies suggest that RAS and IDH2 mutations cooperate to promote leukemogenesis.[4] In IDH2-mutant AML, aberrant production of the oncometabolite 2-hydroxyglutarate (2HG) suppresses the TET2 demethylase leading to a hypermethylation phenotype. Inhibition of TET2 induces epigenetic silencing of key RAS negative regulators (Sprouty family of proteins).[4] Enasidenib promotes demethylation to restore expression of RAS negative regulators. Meanwhile, MEK inhibitors were shown to suppress RAS-signaling. Co-targeting of IDH2 and MEK in mice with IDH2 /NRAS-dependent AML resulted in greater improvement in splenomegaly and cytopenia than either drug alone. Thus, we designed a phase 1 study to evaluate combined targeting of IDH2 and MEK in patients with R/R AML. Study Design and Methods: This is an open-label, multi-center, phase 1b study evaluating the safety of the combination of MEK inhibitor cobimetinib and IDH2 inhibitor enasidenib in patients with R/R AML and RAS-pathway mutations. The study will consist of 1) a dose escalation cohort to determine the maximum tolerated dose and the recommended phase 2 dose (RP2D) and 2) a dose expansion cohort to better characterize safety of the combination as well as preliminary efficacy, both in patients with R/R AML. Key eligibility criteria include R/R AML or newly diagnosed AML not eligible for chemotherapy; presence of co-occurring IDH2 and either NRAS, KRAS, PTPN11, NF1, or CBL mutations. The study will evaluate two dose levels of cobimetinib 40mg (DL1) or 60mg (DL2) daily on days 1-21 of a 28 day cycle and the FDA approved dose of enasidenib 100mg daily. The study primary endpoint is safety. Dose-limiting toxicities will be defined during the first cycle of therapy (28 days) as ≥ grade 3 toxicities according to the NCI CTCAE version 5.0. Dose escalation will occur according to a 3+3 design. Secondary endpoints will include overall response (CR, CRi, MLFS, and PR) by ELN 2022 response criteria, median to first response, response duration, median and 1-year OS and EFS, pharmacokinetics, and pharmacodynamics (phospho-ERK by CYTOF). Correlative objectives include determining the expression of RAS negative regulators by RNA sequencing and epigenetic regulation of RAS regulators by bisulfite sequencing and H3K4me3 and H3K27Ac ChIP-Seq. The study will accrue at the City of Hope National Medical Center and Memorial Sloan Kettering Cancer Center. This trial is registered on clinicaltrials.gov: NCT04243785 1. Amatangelo MD, Quek L, Shih A, et al. Enasidenib induces acute myeloid leukemia cell differentiation to promote clinical response. Blood. 2017 Aug 10;130(6):732-741. 2. Stein EM, DiNardo CD, Pollyea DA, et al. Enasidenib in mutant IDH2 relapsed or refractory acute myeloid leukemia. Blood. 2017 Aug 10;130(6):722-731. 3. Wang F, Morita K, DiNardo CD, et al. Leukemia stemness and co-occurring mutations drive resistance to IDH inhibitors in acute myeloid leukemia. Nature Communications. 2021 2021/05/10;12(1):2607. 4. Kunimoto H, Meydan C, Nazir A, et al. Cooperative Epigenetic Remodeling by TET2 Loss and NRAS Mutation Drives Myeloid Transformation and MEK Inhibitor Sensitivity. Cancer cell. 2018 Jan 8;33(1):44-59.e8. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PU.1-Dependent Enhancer Inhibition Separates Tet2-Deficient Hematopoiesis from Malignant Transformation.

We identify moderately impaired PU.1 mRNA expression as a biological modality predisposing Tet2-deficient hematopoietic stem and progenitor cells to malignant transformation. Our study furthermore uncovers a methylation-sensitive PU.1 gene network as a common feature of myeloid leukemia potentially allowing for the identification of patients at risk for malignant transformation. See related commentary by Schleicher and Pietras, p. 378. This article is highlighted in the In This Issue feature, p. 369.

Cystine uptake inhibition potentiates front-line therapies in acute myeloid leukemia

By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.

Promyelocytic leukemia protein (PML) and stem cells: from cancer to pluripotency

The promyelocytic leukemia protein (PML) is the core organizer of cognate nuclear bodies (PML-NBs). Through physical interaction or modification of diverse protein clients, PML-NBs regulate a multitude of - often antithetical- biological processes such as antiviral and stress response, inhibition of cell proliferation and autophagy, and promotion of apoptosis or senescence. Although PML was originally recognized as a tumor-suppressive factor, more recent studies have revealed a "double-faced" agent role for PML. Indeed, PML displayed tumor cell pro-survival and pro-migratory functions via inhibition of migration suppressing molecules or promotion of transforming growth factor beta (TGF-β) mediated Epithelial-Mesenchymal Transition (EMT) that may promote cancer cell dissemination. In this line, PML was found to correlate with poor patient prognosis in distinct tumor contexts. Furthermore, in the last decade, a number of publications have implicated PML in the physiology of normal or cancer stem cells (CSCs). Promyelocytic leukemia protein activates fatty acid oxidation (FAO), a metabolic mechanism required for the asymmetric divisions and maintenance of hematopoietic stem cells (HSCs). In embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), PML is required for maintenance of the naïve and acquisition of the induced pluripotency state, respectively. Correspondingly, PML ablation causes significant morphological gene expression and lineage choice changes. In this review, we focus on the mechanisms orchestrated by PML and PML-NBs in cancer and healthy stem cells, from cell physiology to the regulation of chromatin dynamics.

Splenic Red Pulp Macrophages Provide a Secondary Stem Cell Niche for Chronic Myeloid Leukemia Stem Cells

Leukemic stem cells (LSCs) are responsible for initiation, progression and relapse of the disease. Chronic myeloid leukemia (CML) is a classical stem-cell driven malignancy and represents an attractive model to study LSCs. Resistance of LSCs to treatment is mediated by cell-intrinsic characteristics but also by the interactions of LSCs with their microenvironment. LSCs depend on signals from their surrounding niche in the bone marrow (BM) and spleen in order to maintain stem cell characteristics such as quiescence and self-renewal. The BM as a niche for LSCs is well-investigated and several therapeutic targets are under consideration.Splenomegaly is a hallmark of CML. Nevertheless, the role of the spleen on leukemia and LSCs in unknown. Therefore, this project aims to investigate the potential role of the spleen as an independent secondary niche for CML stem cells and its contribution to disease development and progression.In order to study the role of the spleen in CML development and progression, we retrovirally transduced FACS-purified Lineage- Sca-1+ c-Kit+ BM cells (LSKs) with pMSCV-p210BCR/ABL-IRES-GFP and injected transduced LSKs into splenectomized or sham operated mice. Splenectomy prior to CML induction significantly improved survival compared to sham operated controls (median survival 31 vs. 22 days; p=0.0006) with 20 % of splenectomized animals surviving longer than 90 days (endpoint). In addition, splenectomized animals showed a 3.7-fold reduction (p=0.002) in LSC numbers in the BM when analyzed 18 days after CML induction, suggesting that the spleen influences the disease burden in the BM.We next analyzed the leukemic compartment in the BM and spleen and found a significant enrichment of leukemic stem and progenitor cells in the spleen (20 % spleen vs. 10 % BM; % leukemic stem and progenitor cells of total leukemic cells; p=0.01; 19-fold increase in total amount of LSCs in the spleen; p =0.007). To functionally confirm the phenotypic increase in splenic LSCs, we performed secondary transplantations of total leukemic cells from the BM or spleen at limiting dilution into lethally irradiated secondary recipients. The analysis revealed a significantly higher frequency of LSCs in the spleen compared to the BM (1/41'703 vs. 1/432'594; p=0.02).In addition, we determined the localization of LSCs in the spleen by confocal microscopy on optically cleared spleens. Splenic LSCs were exclusively localized within the red pulp and in close proximity to macrophages. It was previously shown that hematopoietic stem cells reside in direct contact with macrophages in the red pulp of the spleen during extramedullary hematopoiesis (Dutta et al., JEM, 2015). Therefore, we depleted macrophages in CML mice using clodronate liposomes. Spleen weight (867mg vs. 249mg; p<0.0001) and numbers of splenic LSCs (23-fold; p=0.001) were significantly reduced in clodronate treated animals. Importantly, clodronate treatment did not affect LSC viability, as indicated by comparable amounts of AnnexinV+ cells in both groups.In summary, we show that the interaction of splenic niche cells (red pulp macrophages) with LSCs results in the generation of a big reservoir of LSCs which contribute to CML progression. Therefore, our study strongly suggest the presence of an independent secondary splenic LSC niche in addition to the well characterized BM LSC niche in CML. DisclosuresNo relevant conflicts of interest to declare.

Dual Sphingosine Kinase and Bcl-2 Inhibition Exhibits Synergistic Cell Death in Acute Myeloid Leukemia

Background:Pro-survival Bcl-2 family proteins such as Mcl-1 and Bcl-2 have garnered significant interest as therapeutic targets due to their ability to suppress the apoptotic potential of pro-apoptotic Bax and Bak. In response to cell stress, BH3-only proteins sequester Bcl-2 family members, promoting apoptosis. Small molecule inhibitors such as the Bcl-2 inhibitor Venetoclax have demonstrated poor single agent efficacy in AML. This poor efficacy is a result of AML cells being highly dependent on the pro-survival Bcl-2 family member Mcl-1. Sphingosine kinase 1 (SK1) is a signalling enzyme with established roles in oncogenesis and has recently emerged as a potential therapeutic target in leukaemia. SK1 promotes oncogenic signalling through the production of sphingosine-1-phopshate and subsequent activation of G-protein coupled receptors. In addition, SK1 has been reported to modulate Mcl-1 expression in a number of haematological malignancies including AML. Thus we sought to investigate the efficacy of targeting SK1 to sensitise AML cells to Venetoclax.Results:To determine how SK1 inhibition influences Mcl-1 levels we utilised a short hairpin RNA (shRNA) approach targeting members of the BH3 only protein family (Bid, Bim, Bad, Puma and Noxa). Targeting of Noxa significantly attenuated the cytotoxic effects of the SK1 inhibitor, MP-A08. Immunoprecipitation of Mcl-1 in response to MP-A08 treatment revealed increased association of BH3 only protein, Noxa and ubiquitin-ligase FBW7 suggesting this triggers Mcl-1 degradation in agreeance with previous studies. No interaction was observed between Mcl-1 and Bim further supporting the importance of Noxa in MP-A08 induced Mcl-1 degradation.Canonical pathway analysis of MP-A08 treated cells has previously shown to enrich for genes associated with the unfolded protein response (UPR) (Powell et al. Blood 2017). Western blot analysis recapitulated these findings with activation of the PERK arm, typified by eIF2a phosphorylation and ATF4 upregulation. ATF4 has previously been shown to bind within the Noxa promoter and drive transcription in response to ER stress (Wang et al PNAS 2009). Maintaining global translation with eIF2B agonist, ISRIB blocked Noxa transcription in response to MP-A08 suggesting ATF4 drives Noxa mediated Mcl-1 degradation.Given the Noxa dependent Mcl-1 degradation in response to MP-A08 treatment, we sought to assess the efficacy of combined treatment with the Bcl-2 inhibitor, Venetoclax. Combinational therapies with sub-cytotoxic doses of MP-A08 and Venetoclax induced synergistic cell death in AML cell lines, primary patient AML blasts and leukemic stem and progenitor cells. Synergy could be observed as early as two hours, in agreeance with Noxa upregulation and binding Mcl-1, implicating this as key for synergistic cell death.Conclusions:We have demonstrated SK1 inhibition invokes a pro-apoptotic UPR response via ATF4 driven Noxa upregulation and Mcl-1 degradation. Nullification of the pro-survival function of Mcl-1 by Noxa renders AML cells vulnerable to Bcl-2 targeting strategies such as Venetoclax providing a novel therapeutic angle to enhance patient response to Venetoclax. Current work is underway assessing MP-A08 and Venetoclax using patient-derived xenograft models. DisclosuresNo relevant conflicts of interest to declare.

The Novel Imipridone ONC212 Induces Pronounced Anti-Leukemia Effects in Vitro and In Vivo and Is Highly Synergistic with the BCL-2 Inhibitor ABT-199

Imipridones constitute a novel class of molecules that includes the first-in-class anti-cancer compound ONC201. ONC201 has shown promising clinical activity against hematological and solid malignancies (Allen et al., Oncotarget, 2016). ONC212 is a second-generation imipridone with higher potency than ONC201. We first confirmed the overall in vitro anti-tumor effects of ONC212 using the collection of 1,088 human cancer cell lines available from the Genomic of Drug Sensitivity in Cancer Project. Ranking the sensitivity of these cancer cell lines to ONC212 by tumor type revealed that leukemias and lymphomas were especially sensitive. In fact, ONC212 exerted prominent apoptogenic effects in AML cell lines with > 20-fold potency compared to ONC201 (ED50s at 72 hr; 6.4 μM vs 258.7 nM, respectively for OCI-AML3 cells). Similar results were observed in primary AML cells, but there was no activity against normal bone marrow (BM) cells. We previously demonstrated that ONC201 induces apoptosis via an atypical integrated stress response (ISR, Ishizawa et al., Sci Signal, 2016). Similarly, ONC212 showed ISR activation accompanying its apoptogenic effects in AML cells which was agnostic to the mutation status of p53. However, because of the higher sensitivity of wild-type p53 cells to ONC212, we speculated that this effect could also reflect a p53-dependent apoptosis mechanism. Indeed, p53 knockdown reduced the ONC212-induced apoptosis in MV4;11 and MOLM13 cells, suggesting both, p53-dependent and -independent, mechanisms.We next investigated the effects of ONC212 in vivo in a systemic AML xenograft model using OCI-AML3 cells expressing luciferase. Biweekly oral administration of 50 mg/kg ONC212 markedly inhibited AML expansion and prolonged overall survival (p = 0.0003). Median survival increased from 43 d in controls to 49 d in the ONC212-treated group (+14%). For in vivo functional assessment of ONC212's anti-tumor effects against leukemia stem and progenitor cells, we utilized our patient-derived xenograft (PDX) mouse model. We treated primary PDX cells with ONC212 (250 nM, 36 hr) ex vivo, and then injected identical numbers of viable cells (treated or untreated) into recipient NSG mice. After one month, the percentages of human CD45+ cells in the peripheral blood, spleen, and BM were significantly decreased in the ONC212-treated group. The median survival in this group was remarkably different: 82 d compared to 36 d for controls (+128%, p<0.0001). These results indicated that ONC212 targets AML stem cells with potential to eradicate the disease.Because BCL-2 is generally considered protective against ISR-mediated apoptosis and ONC212 induced p53-mediated apoptosis as demonstrated above, we hypothesized that the BCL-2 inhibitor ABT-199 or the p53 activator nutlin-3a could further sensitize AML cells to ONC212. Indeed, the in vitro combination of ONC212 plus ABT-199 or nutlin-3a synergistically induced apoptosis in AML cells. Furthermore, the combination of ONC212 and ABT-199 showed highly significant synergistic anti-leukemia effects in vivo in a MOLM-13 xenograft mouse model. The combinatorial treatment prolonged overall median survival from 20 d for untreated controls to 21 d for each agent as monotherapy to 30 d for the combination.Since the G-protein-coupled receptor (GPCR) dopamine receptor D2 is the putative target of first-generation imipridone ONC201, we performed a PathHunter β-arrestin screening to determine if ONC212 also bound GPCRs. ONC212 did not engage DRD2 but instead specifically engaged the orphan GPCR GPR132, which is a putative tumor suppressor when activated. Interestingly, expression of GPR132 mRNA was highly correlated with sensitivity to ONC212. Furthermore, GPR132 overexpression induced cell death and ONC212 treatment induced GPR132 mRNA expression in AML cell lines, suggesting that GPR132 could be a potential therapeutic target in hematological malignancies.Taken together, ONC212, and its putative target GPR132, represent a promising novel anti-cancer strategy for AML therapy that warrants further development. DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Stogniew:Oncoceutics: Employment. Andreeff:Daiichi Sankyo: Consultancy.

Leukemia stemness and co-occurring mutations drive resistance to IDH inhibitors in acute myeloid leukemia

Allosteric inhibitors of mutant IDH1 or IDH2 induce terminal differentiation of the mutant leukemic blasts and provide durable clinical responses in approximately 40% of acute myeloid leukemia (AML) patients with the mutations. However, primary resistance and acquired resistance to the drugs are major clinical issues. To understand the molecular underpinnings of clinical resistance to IDH inhibitors (IDHi), we perform multipronged genomic analyses (DNA sequencing, RNA sequencing and cytosine methylation profiling) in longitudinally collected specimens from 60 IDH1- or IDH2-mutant AML patients treated with the inhibitors. The analysis reveals that leukemia stemness is a major driver of primary resistance to IDHi, whereas selection of mutations in RUNX1/CEBPA or RAS-RTK pathway genes is the main driver of acquired resistance to IDHi, along with BCOR, homologous IDH gene, and TET2. These data suggest that targeting stemness and certain high-risk co-occurring mutations may overcome resistance to IDHi in AML.

Abstract IA18: The PI3K isoforms in myeloid leukemia and hematopoietic stem cells

Abstract The Class IA PI3 kinase isoforms (p110α, β, and δ) transduce many growth factor signals that are important for the proliferation, differentiation, and self-renewal of hematopoietic stem cells (HSCs). Mutations in growth factor receptors or RAS proteins are commonly observed in patients with acute myeloid leukemia (AML), leading to activation of the PI3K/AKT pathway. Therefore, PI3K inhibition is an attractive therapeutic strategy for a large subset of AML patients. We previously reported that p110α is dispensable for HSC function, suggesting that redundancy exists between the Class IA isoforms in HSCs (Gritsman et al., J Clin Invest 2014). However, we have identified a specific role for p110α in RAS-mutated myeloid leukemia. Furthermore, we found that the p110α-selective inhibitor BYL-719 can sensitize RAS-mutated leukemic cells to the MEK inhibitor MEK-162. While PI3K inhibitors have multiple clinical indications, including in hematologic malignancies, it is still unclear whether PI3K plays an important role in normal HSC function. Upon environmental stresses like chemotherapy or infection, quiescent HSCs must enter the cell cycle to re-establish homeostasis and promote emergency myelopoiesis. To test for redundancy between p110α and p110δ in HSCs, we generated p110α and p110δ double knockout (DKO) mice, with germline deletion of p110δ and conditional deletion of p110α. DKO mice have leukopenia, anemia, and decreased numbers of lymphoid-primed multipotent progenitors (LMPPs) in the bone marrow, though HSC numbers are unchanged. In competitive repopulation assays, B-cell maintenance was severely impaired, while the myeloid and T-cell lineages were relatively preserved. This suggests that HSC function is not impaired at steady state. We performed transcriptome analysis of DKO, p110δ, and p110α KO HSCs and LMPPs after bone marrow transplantation. Gene set enrichment analysis revealed downregulation of gene sets associated with cell cycle progression. Interestingly, we also observed negative enrichment of inflammatory response gene sets in both p110δ KO and DKO HSCs and LMPPs. To examine the roles of p110δ and α in the hematopoietic stress response, we injected p110δ, DKO, and WT mice with 5-fluorouracil. We observed significantly decreased survival of 5-FU-treated DKO mice due to impaired hematopoietic recovery, associated with the failure of HSCs to enter the cell cycle. Consistent with this finding, we also observed a decrease in myeloid reconstitution by DKO HSCs in secondary competitive transplantation, suggesting that p110α and p110δ play redundant roles in emergency myelopoiesis. We also observed defects in downstream signaling in DKO HSCs and progenitors in response to IL1β or TNFα stimulation. Our results suggest that p110α and δ act in a redundant fashion to transduce specific inflammatory signals in HSPCs in response to hematopoietic stress. Our results have important implications for the use of PI3K inhibitors in combination with chemotherapy and in other hematopoietic stress conditions. Citation Format: Shayda Hemmati, Taneisha Sinclair, Meng Tong, Boris Bartholdy, Rachel Okabe, Kristina Ames, Leanne Ostrodka, Tamanna Haque, Imit Kaur, Anupriya Agarwal, Jean Zhao, Thomas Roberts, Kira Gritsman. The PI3K isoforms in myeloid leukemia and hematopoietic stem cells [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 IA18.

CULMINATE: A phase II study of cusatuzumab + azacitidine in patients with newly diagnosed AML, ineligible for intensive chemotherapy.

TPS7565 Background: AML, the most common acute leukemia in adults, is a heterogeneous malignancy characterized by uncontrolled clonal expansion of hematopoietic progenitor cells. Median diagnosis age is ~67 yrs. Despite current therapies prognosis is poor with 5-yr OS ~25% for patients (pts) ≥65 yrs. For pts unable to receive intensive chemotherapy, survival rates are worse, indicating a critical need to develop better treatments. CD70 is expressed on &gt;95% of AML blasts harvested from newly diagnosed AML pts but not on normal hematopoietic stem cells nor most normal tissues. Cusatuzumab is a first-in-class, high-affinity anti-CD70 monoclonal antibody with multiple mechanisms of action, including Fc-mediated cytotoxicity with enhanced ADCC and inhibition of CD70/CD27 signaling, resulting in leukemia blast and stem cell cytotoxicity. As cusatuzumab and azacitidine target distinct pathways of myeloblast propagation, a combination may have a synergistic therapeutic effect and overcome treatment resistance. Initial data from a Phase I study (NCT03030612) with cusatuzumab (1–20 mg/kg) + standard dose azacitidine in AML pts ineligible for intensive chemotherapy showed no dose-limiting toxicity and a CR/CRi (CR with partial/incomplete hematologic recovery) in 10 of 12 pts (ASH 2019, Abs #234). This abstract describes a follow-on Phase II study (NCT04023526). Methods: CULMINATE is a 2-part study of cusatuzumab + azacitidine to determine the optimal dose of cusatuzumab (Table). Inclusion criteria: ≥18 yrs with de novo or secondary AML unfit for intensive therapy (≥75 or &lt;75 yrs with a comorbidity [i.e. ≥1 of: ECOG 2, severe cardiac/pulmonary or moderate hepatic impairment]). In Part 1, pts are randomized 1:1 to cusatuzumab 10 or 20 mg/kg (IV, on Days 3 and 17 of each 28-day cycle) + azacitidine (75 mg/m2 SC or IV on Days 1–7). Data will be reviewed after 15, 30 and 50 pts are enrolled into each arm to select the cusatuzumab dose for the Part 2 expansion cohort in which efficacy and safety will be further evaluated. Follow-up continues until death, loss to follow-up or study end. The primary objective is to determine CR rate. Secondary objectives include rate of CRi/CRh, rate of MRD-negativity, ORR, time to and duration of response, pharmacokinetics, immunogenicity, transfusion independence and safety. Enrollment began in Sept 2019 and is currently two-thirds complete. Clinical trial information: NCT04023526 . [Table: see text]

Cancer testis antigen Cyclin A1 harbors several HLA-A*02:01-restricted T cell epitopes, which are presented and recognized in vivo

Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8+ T cells from two healthy donors with single-peptide-pulsed dendritic cells or monocytes. Intracellular cytokine staining quantified the enrichment of peptide-specific functional T cells. Seven peptides were immunogenic, three of them against both donors. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA-A*02:01-positive AML cell line THP-1. Immunopeptidome analysis based on direct isolation of HLA-presented peptides by mass spectrometry of primary AML and OC samples identified four naturally presented epitopes of Cyclin A1. The immunopeptidome of HeLa cells transfected with Cyclin A1 and HLA-A*02:01 revealed six Cyclin A1-derived HLA ligands. Epitope p410–420 showed high affinity to HLA-A*02:01 and immunogenicity in both donors. It proved to be naturally presented on primary AML blast and provoked spontaneous functional response of T cells from treatment naïve OC and, therefore, warrants further development for clinical application.