Activity In Acute Myeloid Leukemia Research Articles

BP1223, a Novel T Cell Engager Targeting CD39 for Potent Antitumor Activity in Acute Myeloid Leukemia

BP1223, a Novel T Cell Engager Targeting CD39 for Potent Antitumor Activity in Acute Myeloid Leukemia

Isolation, structural characterization of the first disintegrin from Bothrops moojeni venom and its antitumor activity in acute myeloid leukemia

Isolation, structural characterization of the first disintegrin from Bothrops moojeni venom and its antitumor activity in acute myeloid leukemia

WT1 together with RUNX1::RUNX1T1 targets DUSP6 to dampen ERK activity in acute myeloid leukaemia.

Wilms' tumour 1 (WT1) can function as an oncogene or a tumour suppressor. Our previous clinical cohort studies showed that low WT1 expression at diagnosis independently predicted poor outcomes in acute myeloid leukaemia (AML) with RUNX1::RUNX1T1, whereas it had an opposite role in AML with non-favourable cytogenetic risk (RUNX1::RUNX1T1-deficient). The molecular mechanism by which RUNX1::RUNX1T1 affects the prognostic significance of WT1 in AML remains unknown. In the present study, first we validated the prognostic significance of WT1 expression in AML. Then by using the established transfected cell lines and xenograft tumour model, we found that WT1 suppresses proliferation and enhances effect of cytarabine in RUNX1::RUNX1T1(+) AML but has opposite functions in AML cells without RUNX1::RUNX1T1. Furthermore, as a transcription factor, WT1 physically interacts with RUNX1::RUNX1T1 and acts as a co-factor together with RUNX1::RUNX1T1 to activate the expression of its target gene DUSP6 to dampen extracellular signal-regulated kinase (ERK) activity. When RUNX1::RUNX1T1-deficient, WT1 can activate the mitogen-activated extracellular signal-regulated kinase/ERK axis but not through targeting DUSP6. These results provide a mechanism by which WT1 together with RUNX1::RUNX1T1 suppresses cell proliferation through WT1/DUSP6/ERK axis in AML. The current study provides an explanation for the controversial prognostic significance of WT1 expression in AML patients.

Moojecin: The first disintegrin from Bothrops moojeni venom and its antitumor activity in acute myeloid leukemia

Disintegrins are a class of peptides found in snake venom that inhibit the activity of integrins, which are essential cell adhesion receptors in tumor progression and development. In this work, moojecin, a RGD disintegrin, was isolated from Bothrops moojeni snake venom, and its antitumor potential in acute myeloid leukemia (AML) HL-60 and THP-1 cells was characterized. The isolation was performed using a C18 reverse-phase column in two chromatographic steps, and its molecular mass is 7417.84 Da. N-terminal and de novo sequencing was performed to identify moojecin. Moojecin did not show cytotoxic or antiproliferative activity in THP-1 and HL-60 at tested concentrations, but it exhibited significant antimigratory activity in both cell lines, as well as inhibition of angiogenesis in the tube formation assay on Matrigel in a dose-dependent manner. A stronger interaction with integrin αVβ3 was shown in integrin interaction assays compared to α5β1, and the platelet aggregation assay indicated an IC50 of 5.039 μg/mL. Preliminary evaluation of disintegrin toxicity revealed no incidence of hemolysis or cytotoxic effects on peripheral blood mononuclear cells (PBMCs) across the tested concentrations. Thus, this is the first study to report the isolation, functional and structural characterization of a disintegrin from B. moojeni venom and bring a new perspective to assist in AML treatment.

Abstract 4181: AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in acute myeloid leukemia

Abstract Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross-reactive for both mouse and human AXL protein, to non-invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold prediction software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti-tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross-reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre-injection of cold sdAb20-Fc was required to maximize the tumor-to-background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients. Citation Format: Niels Vandewalle, Hatice Satilmis, Emma M. Verheye, Rong Fan, Yanmeng Wang, Timo W. De Groof, Tessa Kerre, Nathan De Beule, Ann De Becker, Elke De Bruyne, Eline Menu, Karin Vanderkerken, Karine Breckpot, Nick Devoogdt, Kim De Veirman. AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4181.

AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in Acute Myeloid Leukemia.

Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross-reactive for both mouse and human AXL protein, to non-invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold2 software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti-tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross-reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre-injection of cold sdAb20-Fc was required to maximize the tumor-to-background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients.

Discovery of YSR734: A Covalent HDAC Inhibitor with Cellular Activity in Acute Myeloid Leukemia and Duchenne Muscular Dystrophy.

Histone deacetylases (HDACs) have emerged as powerful epigenetic modifiers of histone/non-histone proteins via catalyzing the deacetylation of ε-N-acetyl lysines. The dysregulated activity of these Zn2+-dependent hydrolases has been broadly implicated in disease, notably cancer. Clinically, the recurring dose-limiting toxicities of first-generation HDACi sparked a paradigm shift toward safer isoform-specific molecules. With pervasive roles in aggressive diseases, there remains a need for novel approaches to target these enzymes. Herein, we report the discovery of YSR734, a first-in-class covalent HDACi, with a 2-aminobenzanilide Zn2+ chelate and a pentafluorobenzenesulfonamide electrophile. This class I selective proof of concept modified HDAC2Cys274 (catalytic domain), with nM potency against HDAC1-3, sub-μM activity in MV4-11 cells, and limited cytotoxicity in MRC-9 fibroblasts. In C2C12 myoblasts, YSR734 activated muscle-specific biomarkers myogenin/Cav3, causing potent differentiation into myotubes (applications in Duchenne Muscular Dystrophy). Current efforts are focused on improving in vivo ADME toward a preclinical covalent HDACi.

Mediator Kinase/CDK8 Inhibition As a Strategy to Improve FLT3 Inhibitor Activity in Acute Myeloid Leukemia

Acquired resistance to targeted FLT3 tyrosine kinase inhibitors (TKIs) remains a major barrier to durable clinical responses for patients with FLT3-mutant AML. Multiple resistance mechanisms to next generation FLT3 TKIs converge upon RAS/MAPK pathway activation, including protective cytokine stimuli of the bone marrow microenvironment or through secondary oncogenic RAS mutations. RAS mutations also decrease efficacy of the BCL2 inhibitor venetoclax now widely used in AML, suggesting an anti-apoptotic influence of MAPK signaling. These findings highlight a significant unmet need for novel therapeutic combinations to address MAPK-mediated resistance in AML. We report results from a genome-wide CRISPR interference (CRISPRi) apoptosis screen using FLT3-ITD mutant MOLM-14 cells treated with the FLT3 TKI gilteritinib while cultured in HS5 conditioned media (CM) to mimic cell-extrinsic MAPK-driven TKI resistance. We identified RNA pol II and Mediator kinase/CDK8 ascritical nodes for AML cell survival in the setting of FLT3 inhibition. We further demonstrated that CDK8 inhibition with the small-molecule SEL120 re-sensitized multiple FLT3-mutant AML cell lines and primary samples to gilteritinib. We also performed a second CRISPRi apoptosis screen to identify hits that sensitize to gilteritinib in a model of cell-intrinsic MAPK-mediated resistance using MOLM-14 cells that harbor secondary NRAS-G12C mutation.We used Enrichr to perform an integrated analysis and validated the Core Mediator Complex and CDK8 knockdown genes as cellular components significantly enriched among sensitizing hits in both screens. The Mediator subunit MED12 was identified as a top gene level hit that strongly sensitized cells to gilteritinib in both screens, further emphasizing that Mediator kinase and its transcriptional regulation may be a central pathway engaged in MAPK-dependent survival in FLT3-mutant AML. To better understand potential mechanisms of increased combinatorial CDK8i/FLT3i activity, we performed RNA-seq in MOLM-14 cells treated with gilteritinib, SEL120, or the combination in HS5 CM. Using GSEA we found that gilteritinib stimulated adaptive interferon/inflammatory gene signatures at 16h drug treatment, but this response was restrained by addition of SEL120. Additionally, we identified a striking upregulation of the lineage-controlling transcription factor IRF8 and its gene expression signature with concomitant silencing of SPI1 transcriptional output in cells treated with gilteritinib/SEL120. CDK8i treatment alone in AML has previously been shown to increase expression of super-enhancer (SE) associated genes, including IRF8. Interestingly, SPI1was also a significant gene-level hit in both CRISPRi screens. Using CRISPRi we repressed IRF8 expression in MOLM-14, MOLM-14 NRAS-G12C, and MV411 (another FLT3-mutant line) cells and found this significantly blunted the combinatorial pro-apoptotic effect of SEL120, implying that induction of IRF8 expression plays a significant role in driving response to combined gilteritinib/SEL120. We finally assessed in vivo activity of gilteritinib combined with SEL120. We first performed pilot toxicity studies in NSG mice and determined that gilteritinib 30 mg/kg, SEL120 30 mg/kg, or the combination dosed 5 days/week by oral gavage (OG) were regimens well tolerated by mice. We then engrafted luciferase-tagged MOLM-14 and MOLM-14 NRAS-G12C cells into NSG mice and treated for 4-weeks with gilteritinib, SEL120, or the combination. We found gilteritinib/SEL120 extended survival in both MOLM-14 and MOLM-14 NRAS-G12C models. Similar efficacy studies are ongoing in FLT3-mutant patient-derived xenograft (PDX) models with NSGS mice and will be reported. Our results indicate that FLT3-mutant AML cells employ an adaptive inflammatory response to evade FLT3i-induced apoptosis, and CDK8i represses this adaptation while concomitantly increasing the ratio of IRF8 to SPI1-driven gene expression programs. We speculate this latter effect may in part alter an AML cell's differentiation state to sensitize cells to apoptosis, though further study to explore this hypothesis is needed. Our in vitro and in vivo efficacy data further validated combined FLT3i/CDK8i as a promising investigational strategy to pre-empt or overcome MAPK-mediated FLT3 TKI resistance.

Phase 1b/2 Study of Escalating Doses of the NEDD8 Activating Enzyme Inhibitor Pevonedistat Administered in Combination with Standard Induction Therapy (Cytarabine and Idarubicin) in Newly Diagnosed High Risk Acute Myeloid Leukemia

Introduction: Pevonedistat is a potent and selective small-molecule inhibitor of the NEDD8-activating enzyme that has demonstrated clinical activity in acute myeloid leukemia (AML). Preclinical studies suggest that pevonedistat synergistically enhances the activity of cytarabine in AML. We investigated the safety and efficacy of pevonedistat in combination with 7+3 cytarabine and idarubicin induction chemotherapy in adults with high-risk AML. Methods: This open-label, multicenter phase 1b/2 study (NCT03330821) enrolled patients aged ≥18 years with newly diagnosed high-risk AML, defined as therapy-related AML, AML with antecedent myelodysplastic syndrome or AML with myelodysplasia-related changes, the presence of adverse genetic features per ELN 2017 guidelines, or being age ≥55. Eligible patients had an ECOG status of 0-2. Patients were administered pevonedistat on days 1, 3, and 5 of each 28-day cycle in combination with cytarabine 100 mg/m 2 on days 1-7 and idarubicin 12 mg/m 2 on days 1-3. In the phase 1b dose-escalation part of the study, a standard 3+3 design was used to evaluate 2 dose levels of pevonedistat: 15 and 20 mg/m 2. Consolidation consisted of up to 4 cycles of cytarabine (1-3 g/m 2 q12h) on days 1, 3, and 5 or transplant. For pharmacodynamic (PD) assessment, RT-PCR assays were performed on peripheral blood samples to measure expression levels of 7 NRF-2 target genes (ATF3, GCLM, GSR, NQ01, SLC7A11, SRXN1, and TXNRD1). The primary objective for the phase 1b study was to determine the RP2D of pevonedistat in combination with cytarabine and idarubicin. The primary objective for the phase 2 study was to determine the composite complete response rate (CRc; CR+CRi). Secondary objectives included relapse-free survival (RFS), overall survival (OS), and safety and tolerability. Results: A total of 28 patients were enrolled at 5 sites in the US between August 2018 and August 2021. Six patients were enrolled in the phase 1b study and 22 patients in phase 2. Baseline patient characteristics are shown in Table 1. The median age was 61 years (range 47-74). Per the 2017 ELN risk classification, 8 of 28 patients (29%) had favorable risk, 8 (29%) had intermediate risk, and 12 (43%) had adverse risk. All 28 patients were included for safety and response assessments. In phase 1b, none of the 6 patients experienced dose-limiting toxicity at 15 or 20 mg/m 2. Thus, the RP2D of pevonedistat was determined to be 20 mg/m 2. The CRc rate was 64% (95% CI 44-81) with 17 patients achieving CR and 1 patient achieving CRi. Of the 18 patients who achieved CR or CRi, 16 (89%) achieved MRD negativity by multiparameter flow cytometry, and 7 (39%) proceeded to HSCT. The CRc rates for patients in the ELN 2017 favorable, intermediate, and adverse risk groups were 100% (95% CI 63-100), 63% (95% CI 29-89), and 42% (95% CI 18-71), respectively. Eight patients were included in the interim PD analysis. Among patients who achieved CR or CRi (n=6), 4 (ATF3, GSR, SLC7A11, and SRXN1) of the 7 NRF-2 target genes measured were significantly upregulated at 5 hours post-pevonedistat induction compared to baseline. There was no significant increase in NRF-2-regulated gene transcript levels among patients with refractory disease (n=2). The median follow-up was 31.1 months (range 19.1-55.9). The median RFS was 24.4 months (95% CI 7-54.3+) and median OS was 25 months (95% CI 11.9-55.9+). The estimated 1-year and 2-year RFS were 72% (95% CI 52-93) and 55% (95% CI 23-78), respectively. The estimated 1-year and 2-year OS were 68% (95% CI 51-85) and 53% (95% CI 34-72), respectively. The most common grade ≥3 treatment-related adverse events were anemia (61%), febrile neutropenia (25%), and hypophosphatemia (18%). No treatment-related deaths were observed. Conclusion: Pevonedistat in combination with cytarabine and idarubicin was well tolerated among adults with newly diagnosed, high-risk AML and induced deep remissions with promising RFS and OS.

KDM6A Modulates Anti-Tumor Immune Response By Integrating Immunogenic Cell Death in Human Acute Myeloid Leukemia

Identifying tumor cell-epigenetic regulation of immune activation in acute myeloid leukemia (AML) would be instrumental in developing next-generation of targeted therapy. AML cells have low mutational burden; inefficient ‘cross-presentation’ through conventional dendritic cells (DC) and immune activation are the major challenges for AML immunomodulation. ‘Immunogenic cell death’ (ICD) represents changes in the composition of the tumor cell surface as well as the release of ‘damage associated molecular patterns’ (DAMPs) in the tumor microenvironment that stimulates the dysfunctional immune system. DAMPs in turn operate on a series of receptors expressed by DCs and natural killer (NK) cells and contribute to long-lasting, protective anti-tumor immunity. For example, plasma membrane exposure of endoplasmic-reticulum (ER)-translocated calreticulin (ecto-CRT), which functions as an ‘eat me’ signal, is an important hallmark of ICD. Previous study suggested that calreticulin (CALR) promotes immunity and type I interferon-dependent survival in mice with AML (Fucikova, Blood 2016; Chen, Oncoimmunology 2017). In addition, ecto-CRT on malignant blasts correlated with improved NK cell-mediated cytotoxicity in AML patients with superior overall survival (Truxova, Haematologica 2020). Ecto-CRT has also been identified as an endogenous sterile ligand for NK cells (Sen Santara, Nature 2023). Nevertheless, molecular regulation of ICD in the context of epigenetic derangements and inflammation and its implication in AML immunotherapeutic modulation is poorly understood. KDM6A is a histone 3 lysine 27 demethylase that plays tumor suppressor function in AML (Gozdecka, Nat Genet 2018; Sera, Blood 2021). KDM6A escapes X-chromosome inactivation, and loss-of-function deletion or point mutations associate with resistance to ‘3+7‘ chemotherapy (Stief, Leukemia 2020). We have identified that in comparison to control AML cells, deficiency of KDM6A ( KDM6A-kd) associated with daunorubicin/etoposide-induced ICD, which was manifested by significant increase (2-3 fold , P<0.05) in ecto-CRT, plasma membrane exposure of pE1F2α, HSP70 and HSP90 with extracellular release of HMGB1 and ATP. Similarly, pharmacological inhibition of JmjC catalytic function using GSK-J4 sensitized human AML lines as well as primary AML patient bone marrow-derived CD34 + hematopoietic stem/progenitor cells to ICD. Induction in ICD was accompanied by an elevated type I interferon production that was functionally active in promoting interferon regulated gene expression. Interestingly, treatment with acute recombinant IFN-β caused an increase in activated STAT1 and inflammatory gene expression in KDM6A-kd AML cells compared to control AML. Corroborating these results, gene set enrichment analysis revealed a significant enrichment (NES=1.708, P=0.007) of upregulated type I interferon signatures (n=40) in Kdm6a -/- primary leukemia cells. Re-analysis of ChIP-seq results indicated 1647 Kdm6a occupied genes, which were upregulated upon Kdm6a loss. Gene ontology (GO) analysis of the 1647 genes further suggested an enrichment ( P<0.001) of type I interferon-responsive GO terms, indicating Kdm6a role in interferon signaling. Ecto-CRT level relies on premortem ER stress. In OHSU AML, KDM6A expression correlates with ER stress/unfolded protein response. Mechanistically, quantitative-ChIP and promoter-reporter assays indicated that KDM6A-kd de-repressed ICD induced expression of ATF4 and HSPA5 through chromatin and transcriptional remodeling. In agreement with these findings, AML blasts-healthy immune cells ex vivo co-culture experiments revealed that increase in ICD in KDM6A-kd AML was associated with activation (~2-foldincrease inCD80 +CD86 +, P<0.05) and phagocytic function of DCs. Loss of KDM6A was shown to bestow ‘BRCAness’ (Boila, Leukemia 2023), and PARP inhibition itself can cause ICD and augment tumor immunity. We observed that there was a significant induction in ICD and expression of NKG2D ligands ULBP1, ULBP5 in KDM6A-kd AML cells treated with olaparib. In addition, olaparib and GSK-J4 treatment showed additive effects in ICD and immune activation using primary AML CD34 + cells. Together, we illustrate that KDM6A regulates ICD through epigenetic mechanisms, while KDM6A deficient AML subtypes could be sensitized with immunomodulatory targeted therapy.

Assessment of T-Cell Receptor (TCR) Mediated T-Cell Activation in Acute Myeloid Leukemia at Diagnosis Has Implications for Risk Stratification and Response to Immune Checkpoint Inhibitors

Background Relapse in patients with acute myeloid leukemia (AML) has been, in part, attributed to the ability of leukemic blasts to escape immune surveillance through mechanisms such as MHC class II antigen modulation, alteration of the cytokine milieu, and upregulation of inhibitory and immune checkpoint ligands that promote T-cell suppression. Yet, immune checkpoint inhibitors (ICI) used in AML have yielded variable and unpredictable results. Moreover, evaluation of T-cell function is not part of the diagnostic assessment of patients with AML. Methods To better characterize the T-cell dysfunction in AML, we conducted a prospective study on freshly harvested, bone marrow aspirates from patients with AML at presentation, collected under an Institutional IRB-approved banking protocol over the past two years. Using a multiparameter flow cytometry-based assay, we analyzed T-cell activation potential in response to CD3/CD28 (bead-based), T-cell receptor (TCR) stimulation, both alone (n= 40 AMLs and 10 healthy donors) or in combination with a library of ICI (n=15 AML and 5 healthy donors). We selected OX40 (CD134/TNFRSF4) and ICOS (Inducible T-cell costimulator) as surrogate markers for T-cell activation based on their expression pattern post bead-based stimulation in healthy donors' bone marrows. The ICI library comprised 31 therapeutic antibody inhibitors of known immune checkpoints, signaling pathways, and chemokines reported to be implicated in T-cell suppression in AML and other solid tumors. Results Healthy T-cells displayed the highest surface expression of OX40 and ICOS at 5 days post-bead-stimulation, which was selected as the endpoint for our in vitro analyses. We found that bone marrow-derived T-cells from healthy donors upregulated the expression of the markers ICOS and/or OX40 in at least 20% of T-cells post-stimulation (n= 10, ICOS: median 36.9%, range 20-72%; OX40: median 59.3%, range 21.4 -83%). Overall, AML samples displayed a lower degree of ICOS and OX40 T-cell expression in response to TCR stimulation (n=40, ICOS: median 20.5%, range 4-65%; OX-40: median 30.3%, range 0.3-80%). Based on this result, T-cell activation was defined as >20% CD3+ cells ICOS+/OX-40+ with stimulation, with greater than 2-fold changes from baseline ICOS and OX40 expression. Using this criterion, patients with AML were then classified as activators or non-activators. T-cell activation status correlated with ELN 2022 risk (1) (Figure 1A). For the intermediate risk group, we observed that the activators group had a significantly longer relapse-free survival when compared to the non-activators (Figure 1B, n=10, Mantel-Cox log-rank = 0.02, follow up 400 days at the time of writing). While a similar trend was observed in the favorable and the poor risk groups, it did not reach significance in these ELN categories. To assess if the T-cell activation could be rescued or augmented upon immune checkpoint inhibition, we tested the effect of each of the 31 compounds of the ICI library on the degree of T-cell activation in diagnosis bone marrow aspirates of 15 patients (7 activators and 8 non activators) and in 5 healthy donors. Matched peripheral blood (PB) samples were available for 5 of the 15 patients with AML. All tested normal donors reached the threshold for T-cell activation, which was not significantly affected by ICI treatment. The 7 AML activators behaved similarly to the healthy donors. In 2 of the 8 AML non-activators, selected ICI rescued T-cell activation in absence of TCR stimulation, while in the remaining 6 cases, T-cell activation was not achieved under any condition. Similar, but overall dampened activation trends, were observed in PB samples. Conclusions Consistent with the findings of our previous retrospective study (2), the ongoing prospective analysis of T-cell activation of diagnosis bone marrows of patients with AML suggests that testing TCR-mediated T-cell activation potential at diagnosis may help refine risk stratification of the heterogenous ELN intermediate risk group. AML immune suppression seems to be reversible in a minority of cases with the use of selected ICI inhibitors.

Targeting human mitochondrial NAD(P)+-dependent malic enzyme (ME2) impairs energy metabolism and redox state and exhibits antileukemic activity in acute myeloid leukemia

Acute myeloid leukemia (AML) is a fast-growing and highly fatal blood cancer, and recent research has shown that targeting metabolism may be a promising therapeutic approach for treating AML. One promising target is the human mitochondrial NAD(P)+-dependent malic enzyme (ME2), which is involved in the production of pyruvate and NAD(P)H and the regulation of the NAD+/NADH redox balance. Inhibition of ME2 via silencing ME2 or utilizing its allosteric inhibitor disodium embonate (Na2EA) causes a decrease in pyruvate and NADH, leading to a decrease in producing ATP via cellular respiration and oxidative phosphorylation. ME2 inhibition also decreases NADPH levels, resulting in an increase in reactive oxygen species (ROS) and oxidative stress, which ultimately leads to cellular apoptosis. Additionally, ME2 inhibition reduces pyruvate metabolism and the biosynthetic pathway. ME2 silencing inhibits the growth of xenotransplanted human AML cells, and the allosteric ME2 inhibitor Na2EA demonstrates antileukemic activity against immune-deficient mice with disseminated AML. Both of these effects are a result of impaired energy metabolism in mitochondria. These findings suggest that the targeting ME2 may be an effective strategy for treating AML. Overall, ME2 plays an essential role in energy metabolism of AML cells, and its inhibition may offer a promising approach for AML treatment.

Abstract 1673: Discovery of potent, selective, and orally bioavailable GSPT1 degraders and their pre-clinical anti-tumor activity in acute myeloid leukemia and solid tumors

Abstract Molecular glue degraders (MGDs) of neo-substrates that function by hijacking the CRBN E3 ligase are clinically validated as therapeutics for the treatment of hematological malignancies. Novel MGDs of GSPT1, a translation termination factor, elicit dysregulated protein translation termination and subsequent induction of TP53-independent apoptosis in acute myeloid leukemia (AML) cells through activation of the integrated stress response. In addition, dysregulation of translation termination triggered by GSPT1 degradation appears to be cytotoxic specifically to MYC-driven cancers including solid tumors with translational addiction. Phase 1 clinical trials of three novel GSPT1 MGDs have been recently launched for the treatment of AML and/or MYC-driven solid tumors (NCT02848001, NCT04336982, NCT05546268, and NCT05144334). Herein, we present novel GSPT1 MGDs and their pre-clinical results against AML and solid tumors. Our current lead GSPT1 MGDs induced rapid, potent, and durable degradation of GSPT1, resulting in strong inhibition of tumor cell growth in a variety of cell lines of MYC-driven solid tumor and blood cancer (AML and acute lymphoblastic leukemia). Moreover, in ex-vivo assays using a panel of patient-derived primary AML cells, C-10386 was highly sensitive with EC50s at the nanomolar range. Furthermore, a related analogue (C-14207) showed excellent rodent PK profiles and demonstrated dose-dependent PKPD relationship and excellent efficacy in the NCI-H1155 NSCLC xenograft model. Finally, our current lead compounds showed safer profiles in various normal cells compared to other known GSPT1 degraders. Collectively, we have identified novel GSPT1 MGDs as promising development candidates for the treatment of AML and solid tumors. Citation Format: Min Sung Joo, JaeYung Lee, Eun-Jung Kim, Dong Hyuk Ki, Hunmi Choi, Joonwoo Nam, Jaewoo Park, Keon Wook Kang, Wooseok Han. Discovery of potent, selective, and orally bioavailable GSPT1 degraders and their pre-clinical anti-tumor activity in acute myeloid leukemia and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1673.

622MO An asymmetrical CLL1/CD3 bispecific antibody, ABL602, exhibits CLL1 binding-dependent CD3 binding/activation and antitumor activity in acute myeloid leukemia (AML) mouse model and leukemia blasts from AML patients

C-type lectin-like molecule 1 (CLL1) is highly expressed in acute myeloid leukemia blast cells but not in normal hematopoietic stem cells (HSCs). This limited expression of CLL1 makes it as a promising target for the AML therapy compared with other targets such as CD33 and CD123, which are also expressed in HSCs. Previously we have shown potent CLL1-specific antitumor activity using two AML cell line/models. In this study, we determined whether binding affinity of ABL602 to CD3 can be augmented upon binding to CLL1 and expanded our study to various AML cell lines and blasts from AML patients. To determine CLL1 binding-dependent CD3 binding of ABL602, T cell binding was measured by flow cytometry in the presence of CLL1-spiked beads. ABL602-mediated antitumor activity and T cell activation were tested in CLL1 expressing AML cell lines and AML blasts from patients. In vivo antitumor activity was determined in humanized mice bearing AML tumors. ABL602 (2+1), with its CD3 binding arm sterically hindered by CLL1 binding arm, binds to CD3 with a lower affinity compared with 1+1 format in the absence of CLL1-expressing cells. By incubating T cells with beads spiked with various amounts of CLL1 in the presence of ABL602, we proved that ABL602’s binding affinity to CD3 was increased in a CLL1-dependent manner, reaching comparable level to that of 1+1. ABL602 induced superior tumor cell killing than 1+1 reference antibody JNJ-67571244. Moreover, ABL602 showed a dose-dependent anti-tumor activity with tumor regression in OCIAML2 xenograft and HL-60 orthotopic model. In AML patient blasts, CLL1 was highly expressed and ABL602 exhibited a strong T cell activation and tumor killing activity on CLL1+ AML blasts. This study supports the target-specific T cell activation and tumor killing activity of 2+1 heterodimeric structure of ABL602 on both AML cell lines and patient-originated AML blasts and warrants further pre-clinical development.

Generation of Leukaemia-Derived Dendritic Cells (DCleu) to Improve Anti-Leukaemic Activity in AML: Selection of the Most Efficient Response Modifier Combinations.

Dendritic cells (DC) and leukaemia derived DC (DCleu) are potent stimulators of anti-leukaemic activity in acute myeloid leukaemia (AML) and can be generated from mononuclear cells in vitro following standard DC/DCleu-generating protocols. With respect to future clinical applications though, DC/DCleu-generating protocols specifically designed for application in a whole-blood-(WB)-environment must be established. Therefore, we developed ten new DC/DCleu-generating protocols (kits; Kit-A/-C/-D/-E/-F/-G/-H/-I/-K/-M) for the generation of DC/DCleu from leukaemic WB, containing calcium-ionophore, granulocyte-macrophage-colony-stimulating-factor (GM-CSF), tumour-necrosis-factor-alpha, prostaglandin-E1 (PGE1), prostaglandin-E2 (PGE2) and/or picibanil (OK-432). All protocols were evaluated regarding their performance in generating DC/DCleu using refined classification and/or ranking systems; DC/DCleu were evaluated regarding their performance in stimulating anti-leukaemic activity using a cytotoxicity fluorolysis assay. Overall, we found the new kits capable to generate (mature) DC/DCleu from leukaemic WB. Through refined classification and ranking systems, we were able to select Kit-I (GM-CSF + OK-432), -K (GM-CSF + PGE2) and -M (GM-CSF + PGE1) as the most efficient kits in generating (mature) DC/DCleu, which are further competent to stimulate immunoreactive cells to show an improved anti-leukaemic cytotoxicity as well. This great performance of Kit-I, -K and -M in mediating DC/DCleu-based anti-leukaemic immunity in a WB-environment in vitro constitutes an important and directive step for translating DC/DCleu-based immunotherapy of AML into clinical application.

Immunomodulatory kits generating leukaemia derived dendritic cells do not induce blast proliferation ex vivo: IPO-38 as a novel marker to quantify proliferating blasts in acute myeloid leukaemia

(Leukaemia derived) dendritic cells (DC, DCleu) are potent stimulators of anti-leukaemic activity in acute myeloid leukaemia (AML) and can be generated with immunomodulatory kits containing granulocyte-macrophage-colony-stimulating-factor (GM-CSF), prostaglandin-E1 (PGE1), prostaglandin-E2 (PGE2) and/or picibanil (OK-321). Potential adverse effects initiated through kits, especially the proliferation of blasts, must be ruled out to ensure treatment safety. We quantified proliferating blasts with the proliferation markers CD71 and Ki-67 and the novel proliferation marker IPO-38 before and after kit treatment ex vivo. IPO-38 hereby appeared to be the most sensitive marker; a combination with CD71 may add value when assessing proliferation kinetics. Kit treatment did not or only slightly (<5%) induce blast proliferation in most cases. An induction of blast proliferation was only found in single cases and could be compensated by DCleu-induced anti-leukaemic activity in most times. Overall, we appraise kit treatment to be safe in vivo.

Abstract 2591: Combination of the BCL-2 inhibitor ZN-d5 with the WEE1 inhibitor ZN-c3 shows additive or synergistic anti-tumor activity in acute myeloid leukemia (AML) models

Abstract Background: The Bcl-2 inhibitor venetoclax is approved in combination with hypomethylating agents such as azacitidine for the treatment of newly diagnosed elderly patients with AML; however, most patients eventually relapse, especially those with TP53 mutations who have poor prognosis. Wee1 is a crucial cell cycle checkpoint kinase that regulates the G2/M checkpoint in response to DNA damage, and its inhibition can cause mitotic catastrophe and apoptosis. ZN-d5 and ZN-c3 are highly selective and potent inhibitors of Bcl-2 and Wee1, respectively. We show here that the combination is highly active in preclinical models of AML. Methods: Cell proliferation in tumor cell lines and AML patient’s samples were measured using CellTiter-Glo® (Promega). Relevant markers were measured by Western blotting. Anti-tumor efficacy was determined in cell line derived subcutaneous xenograft models of acute myeloid leukemia (AML) and in an AML patient-derived systemic xenograft (PDX) model. Flow cytometry was used for analysis of the PDX model to measure human AML blast populations. Results: The in vitro combination of ZN-d5 and ZN-c3 was tested in AML cell lines and showed synergistic or additive anti-proliferative activity which was independent of BCL-2 sensitivity in some cases. In addition, ZN-d5 in combination with ZN-c3 had a synergistic anti-tumor activity in vivo in cell line-derived AML models, such as MV4-11 and HL-60. Also, the triple combination of ZN-d5 + ZN-c3 + azacitidine in the HL-60 in vivo model resulted in synergistic anti-tumor efficacy and significant tumor growth inhibition compared to single- and double-agent treatments. The ZN-d5 + ZN-c3 combination was also tested in vitro in 29 AML patient-derived samples. Response to ZN-c3 alone was observed in 23/29 samples (IC50 &amp;lt; 450 nM), independent of TP53 mutation status. Response to ZN-d5 alone was observed in 17/29 samples (IC50 &amp;lt;200 nM). The combination resulted in synergy being observed in 12/29 models and had greater anti-proliferative activity than either single agent alone in all samples including those insensitive to Bcl-2 inhibition (IC50 &amp;gt; 10 μM). Combination of ZN-d5 and ZN-c3 was also highly active in an AML patient-derived PDX model, resulting in a 99.5% decrease in human CD45+ blast population in the bone marrow. Finally, the combination of ZN-d5 and ZN-c3 was active in 3/3 ex vivo experiments of patients derived AML samples who had progressed on venetoclax. Conclusions: These results justify testing the ZN-d5 and ZN-c3 combination or possibly the triple combination including azacitidine in AML patients independently of TP53 status. Due to synergism observed with these combination(s) and activity in samples from patients resistant to venetoclax, activity may be seen in patients with low sensitivity to Bcl-2 inhibitors or even in patients who progressed on venetoclax plus azacitidine. Citation Format: Hooman Izadi, Tiffany Hoang, Noah Ibrahim, Joseph Pinchman, Brant C. Boren, Jianhui Ma, Petrus R. de Jong, Jiali Li, Kevin D. Bunker, Ahmed A. Samatar, Fernando Doñate. Combination of the BCL-2 inhibitor ZN-d5 with the WEE1 inhibitor ZN-c3 shows additive or synergistic anti-tumor activity in acute myeloid leukemia (AML) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2591.

Cinobufagin inhibits proliferation of acute myeloid leukaemia cells by repressing c-Myc pathway-associated genes

Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad, Bufo gargarizans. Bufanolides inhibit Na+/K+ ATPase and have similar effects as cardiac glycosides, such as digitoxin or ouabain derived from toxic herbs. Recently, the anti-cancer effects of bufanolides have gained attention, however the underlying molecular mechanisms remain unclear. Selecting cinobufagin as a candidate anti-leukaemia agent, we here conducted transcriptomic analyses on the effect of cinobufagin on human acute myeloid leukaemia (AML) cell lines, HL60 and Kasumi-1. Flow cytometry analysis showed that cinobufagin induced apoptosis in both cell lines. RNA-sequencing (RNA-seq) of the two cell lines treated with cinobufagin revealed commonly downregulated genes with enrichment in the term “Myc active pathway” according to Gene Ontology (GO) analysis. Gene Set Enrichment Analysis (GSEA) of genes downregulated by cinobufagin also showed “MYC_TARGETS_V2” with the highest normalised enrichment score (NES) in both cell lines. In contrast, hallmarks such as “TNFA_SIGNALING_VIA_NFKB”, “APOPTOSIS”, and “TGF_BETA_SIGNALING” were significantly enriched as upregulated gene sets. Epigenetic analysis using chromatin immunoprecipitation and sequencing (ChIP-seq) confirmed that genes encoding cell death-related signalling molecules were upregulated by gain of H3K27ac, whereas downregulation of c-Myc-related genes was not accompanied by H3K27ac alteration. Cinobufagin is an anti-proliferative natural compound with c-Myc-inhibiting and epigenetic-modulating activity in acute myeloid leukaemia.

Understanding the Notch Signaling Pathway in Acute Myeloid Leukemia Stem Cells: From Hematopoiesis to Neoplasia.

Simple SummaryWe review the state of the art of knowledge regarding the Notch signaling pathway to shed light on the role that this pathway has in hematopoiesis and hematological neoplasia, focusing on acute myeloid leukemia.The Notch signaling pathway is fundamental to early fetal development, but its role in acute myeloid leukemia is still unclear. It is important to elucidate the function that contains Notch, not only in acute myeloid leukemia, but in leukemic stem cells (LSCs). LSCs seem to be the principal cause of patient relapse. This population is in a quiescent state. Signaling pathways that govern this process must be understood to increase the chemosensitivity of this compartment. In this review, we focus on the conserved Notch signaling pathway, and its repercussions in hematopoiesis and hematological neoplasia. We found in the literature both visions regarding Notch activity in acute myeloid leukemia. On one hand, the activation of Notch leads to cell proliferation, on the other hand, the activation of Notch leads to cell cycle arrest. This dilemma requires further experiments to be answered, in order to understand the role of Notch not only in acute myeloid leukemia, but especially in LSCs.

Abstract LBA002: Targeting GSPT1 by a novel cereblon E3 ligase modulator for the treatment of Acute Lymphoblastic Leukemia

Abstract Acute Lymphoblastic Leukemia (ALL), the most common childhood cancer and the second most common acute leukemia in adults, arises from clonal expansion of undifferentiated lymphoid precursor cells in bone marrow. Although the majority of childhood ALL cases harbor clonal genetic alterations of transcription factor (TF) gene mutations or rearrangements, TF alterations remain difficult therapeutic targets. Small molecule induced protein degradation is a novel strategy that can be applied to currently undruggable targets such as TF and fusion oncoproteins. In this paradigm, small molecule degraders (either PROTAC or Molecular Glue (MG)) redirect the cell’s endogenous ubiquitin proteasome system and induce ubiquitination of the target protein or non-native substrate of an E3 ligase (neosubstrate) and its subsequent proteasomal degradation. Recently, the CRBN E3 ligase modulator, CC90009 was reported to show potent antitumor activity in acute myeloid leukemia, leading to the identification of GSPT1 (G1 to S phase transition factor) as a CRBN neosubstrate. These findings suggest the potential of MGs to target unanticipated vulnerabilities in different malignancies. Here, using a structurally diverse and unique set of MGs (Molecular Glue Library (MGL)) with confirmed CRBN binding affinity, we sought to identify novel CRBN modulators through phenotypic and proteomic methods. Our screening of the MGL in a panel of representative acute leukemia cell lines, including the CRLF2-rearranged ALL cell line MHH-CALL-4 identified several active MGs with EC50 &amp;lt;5 μM. A lenalidomide competition assay and MHH-CALL-4 CRBN knock down cells confirmed the CRBN dependency of these MGs. Among these compounds, SJ6986, a thalidomide-driven sulfonamide showed potent cytotoxicity in over 10 ALL cell lines tested in vitro. TMT-MS proteomic analyses identified GSPT1/2 as the primary targets for this compound with high selectivity. We next tested SJ6986 activity in a panel of patient derived ALL xenografts (PDX) harboring rearrangement of IGH-CRLF2, EPOR, ATF7IP-JAK2 ex vivo. All the tested tumors were highly sensitive to SJ6986 with IC50s at nanomolar range. PK analyses in NSG mice indicated rapid absorption and over 80% oral bioavailability for SJ6986. PD studies in an IGH-CRLF2 PDX showed dose-dependent degradation of GSPT1 within 48 hours of treatment. Finally, we examined the antitumor activity of SJ6986 in 6 different PDX representative of high-risk subtypes of ALL including near haploid, low-hypodiploid, CRLF2-rearranged and EPOR-rearranged, in vivo for 28 days. SJ6986 was able to dramatically decrease the tumor burden at 1 mg/kg dose in most of the tumor models. Collectively, these results affirm that SJ6986 is a novel CRBN modulator and a potential therapeutic agent by targeting GSPT1 protein with high selectivity and potency for the treatment of ALL. Citation Format: Fatemeh Keramatnia, Yunchao Chang, Gisele Nishiguchi, Jaeki Min, Charles Mullighan, Marcus Fischer, Zoran Rankovic, Fatemeh Keramatnia. Targeting GSPT1 by a novel cereblon E3 ligase modulator for the treatment of Acute Lymphoblastic Leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA002.