Primary Acute Myeloid Leukemia Research Articles

TP53 Altered Acute Myeloid Leukemia Stem Cells Hyperactivate Nuclear Factor-Kappa B

In acute myeloid leukemia (AML), standard therapies induce complete remission in 50-70% of patients, but the overall two-year survival is less than 20-30% because of high relapse rates. AML with mutations or deletions in TP53 (TP53 altered, TP53Alt) is a poor-risk AML subtype that is largely insensitive to chemotherapy, modern targeted agents, and hematopoietic stem cell transplantation. TP53Alts are seen in approximately 10-20% of AML and confer a particularly poor prognosis decreasing the 1-2-year survival rates to 0-10%. Leukemia stem cells (LSCs), the cells that recapitulate and propagate leukemia, are central drivers of leukemia progression and relapse. Therapeutically targeting LSCs is important to curing AML, but LSCs are poorly proliferative in contrast to bulk AML cells and are less sensitive to standard chemotherapy. Despite a broad literature defining the immunophenotype (the cell surface marker expression profile) and mechanisms of self-renewal of LSCs, the TP53 status has not been reported in most studies of human LSCs. Our goal is to define the immunophenotype and molecular states of primary human TP53Alt LSCs. First, we asked whether the LSC immunophenotype of TP53Alt AML is consistent with that of the previously reported LSC immunophenotype in AML. We sorted primary human TP53Alt AML samples and plated cells from sorted compartments in serial colony forming assays (which serve as an in vitro surrogate for self-renewal). We found that the Lineage-CD34+CD38-compartment is enriched in serial colony forming capacity in four primary human TP53Alt samples tested. In another TP53Alt sample, we found that the Lineage-CD34+CD38any compartment was enriched for serial colony formation. In vivo engraftment studies recapitulated these findings in one of the TP53Alt AML sample we tested. In the two CD34-TP53Alt AML samples studied the lineage-CD123+ compartment was enriched in serial colony forming capacity. Together, these findings suggest that the standard AML LSC markers also identifies the LSC compartment of TP53Alt AML. Next, we used mass cytometry (CyTOF) to define the proliferative and signaling states of human TP53Alt AML samples. Previous work has demonstrated that the LSC compartment of AML is poorly proliferative, relative to the bulk leukemia population. Consistent with these findings, we found low levels of Ki67 (a proliferation marker) expression, in TP53Alt LSCs, relative to bulk leukemia cells, in every primary TP53Alt AML that we profiled (n=7). Notably, we found that TP53Alt LSCs express high levels of pNFkB, p4EBP1, and pSTAT5, suggesting that these signals may mediate self-renewal in TP53Alt AML. Indeed, we found that CD69, an NFkB target gene, is elevated in TP53Alt LSCs and minimally expressed in bulk leukemia cells. Furthermore, we found that Ki67 levels are low in the CD69High subset, suggesting that this is a poorly proliferative subset (consistent with having self-renewal capacity). Finally, we sorted primary samples according to CD69 status and found that CD69marks a subset with enhanced serial colony forming capacity. Together, these findings demonstrate that traditional LSC markers can be used to study TP53Alt LSCs and that NFkB activation is correlated with self-renewal function in these LSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Amsa/ARA-C Followed By TBI 4 Gy or High Dose Chemotherapy Conditioning Regimens in Primary Refractory AML - Significantly Worse Outcomes in Younger Patients Conditioned with 4 Gy TBI

Background: Allogeneic stem cell transplantation (allo-SCT) plays a fundamental role in the management of acute myeloid leukemia (AML) and is generally recommended for patients with primary induction failure. The optimal conditioning regimen in preparation for SCT in this specific difficult contex has been the subject of continued controversy. Specifically, the role of total body irradiation (TBI) or chemotherapy (CT) as part of sequential conditioning, continues to be central to this debate. With improvement in CT delivery and the introduction of intravenous (IV) busulfan, the utilization of TBI has decreased in popularity. This is a retrospective, multi-center, registry-based, European Society of Blood and Marrow Transplantation (EBMT) study which aimed to compare the outcomes of allo-SCT with amsacrine (AMSA) /cytarabine (ARA-C) followed by TBI conditioning to CT-based conditioning and thus determine certain prognostic factors that could potentially aid in the decision to implement TBI into a patient's conditioning regimen in preparation for allo-SCT. Patients and Methods: For this analysis, we identified 466 adult patients (58.8% male, median age: 55 years, range: 18-75) with primary refractory AML (73.6% de novo) transplanted between the years 2001 and 2021 (median: 2013) from matched sibling donors (MSD) or matched unrelated donors (MUD: 59.2%), who had received AMSA and ARA-C conditioning followed by CT or TBI sequential conditioning. In patients with available cytogenetic data, 50% had intermediate cytogenetics and 50% had poor cytogenetics. Karnofsky score at the time of transplant was >90 in 57.8% of the patients. The majority (73.1%) received a combination cyclosporine (CSA) and mycophenolate mofetil (MMF) for graft-versus-host disease (GVHD) prophylaxis and 92.7% underwent in vivo T-cell depletion. Results: Of the 466 eligible patients identified, 237 (50.8%) received sequential CT-based conditioning following high-dose AMSA/ARA-C with busulfan-fludarabine (BuFlu) or busulfan-cyclophosphamide-fludarabine (BuCyFlu) and 229 received sequential TBI-based conditioning following high-dose AMSA/ARA-C with a total of 4 Gy and cyclophosphamide-fludarabine (CyFlu). In univariate analysis, there was a significant difference in age between the two groups whereby patients receiving CT-based sequential conditioning were older (59.9 years vs 49.7 years, p<0.0001). Moreover, there was a significant difference in the proportions of MSD/MUD between the two groups with 67.9% being allografted from MUD in the CT group compared to 50.2% in the TBI group (p=0.0001). Given the significant interaction between age and conditioning (sequential TBI vs. CT), we stratified our sample by median age (<55 vs >= 55). In patients younger than 55 years, 2-year leukemia-free survival (LFS) was significantly higher in the CT group (51.9% vs 25.1%, p=0.002), as was overall survival (OS) (57.5% vs 38.3%, p=0.021). CT-based sequential conditioning was also associated with higher rates of grades II-IV and III-IV acute GVHD (38.1% vs 24.7%, p=0.02 and 19.8% vs 8.9%, p=0.022). In multivariate analysis, the use of TBI- vs CT-based sequential conditioning significantly influenced relapse (hazard ratio [HR] 1.98, p=0.018), non-relapse mortality (NRM: HR 2.7, p=0.038), LFS (HR 2.03, p=0.004), OS (HR 1.86 p=0.016) and acute GVHD II-IV (HR 0.45, p=0.014). Moreover, age (per 10 years) significantly impacted NRM (HR 2.42, p=0.0005), female to male donor impacted NRM (HR 2.41, p=0.032) and chronic GVHD incidence (HR 2.21, p=0.033) and in vivo T-cell depletion (TCD) impacted chronic GVHD (HR 0.32, p=0.03). In patients 55 years or older, there was no significant difference in post-allo-SCT outcome between patients who received TBI-based vs CT-based sequential conditioning. Conclusion: The above results highlight a significant difference in outcomes in AML patients receiving sequential TBI-based vs CT-based conditioning in preparation for allo-SCT according to age, whereby patients younger than 55 years had significantly worse outcomes after receiving TBI. This was not reflected in patients aged 55 years or more. Although only 4 Gy, these results emphasize the importance of age in the transplant physician's decision to incorporate TBI into conditioning regimens to optimize outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation from Different Donor Types in Primary Refractory Acute Myeloid Leukemia: A Report from the ALWP of the EBMT

Background Primary refractory acute myeloid leukemia (prAML) is a poor prognosis disease for which allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option currently available. We aimed to estimate outcomes in the recent period, to analyze how different donor types may have impacted survival outcomes and to identify the most relevant predictive factors for allo-HSCT in prAML. Methods We conducted a retrospective multicenter study of adult patients reported to the European Society for Blood and Marrow Transplantation (EBMT) registry who received an allo-HSCT between 2015 and 2020 for prAML. We considered HSCT performed from matched sibling donors (MSD), 10/10 unrelated donors (UD 10/10), 9/10 unrelated donors (UD 9/10) and haploidentical donors. Primary endpoint was leukemia-free survival (LFS), secondary endpoints were overall survival (OS), relapse incidence (RI) and non-relapse mortality (NRM). A Cox proportional hazards model was used for multivariate analysis. Results We included 1574 patients. Median age was 56.9 years (range 18.1-77.9). Cytogenetic risk was intermediate in 855 (54.3%), adverse in 679 (43.2%) and favorable in 40 (2.5%) patients. Donor type was distributed as follows: MSD n = 521 (33.1%), UD 10/10 n = 540 (34.3%), UD 9/10 n = 153 (9.7%), haploidentical n = 360 (22.9%). Table 1 summarizes patient and transplant characteristics. Patients allografted from an MSD were younger, allografted faster after diagnosis, more often received myeloablative conditioning (MAC) (details in Table 1) and had less secondary AML. Use of post-transplant cyclophosphamide, and bone marrow as a stem cell source were more frequent in haploidentical transplants. LFS at 2 years was significantly lower in haploidentical transplants (27% [95%CI: 22-32.2]) compared to MSD (35.2% [95%CI: 30.6-39.9]), to UD 10/10 (37.6% [95%CI: 33.2-42.1]) and to UD 9/10 (35.2% [95%CI: 27.1-43.5]) (p=0.003). Haploidentical transplants also showed inferior OS at 2 years: 31.2% (95%CI: 25.8-36.8) compared to 40.9% (95%CI: 35.9-45.8) in MSD, to 45.7% (95%CI: 41.1-50.2) in UD 10/10 and to 43% (95%CI: 34.5-51.3) in UD 9/10 (p=0.001). In the UD 9/10 group we observed the lowest RI (33.5% [95%CI: 25.6-41.6], 46.7% [95%CI: 41.8-51.4] in MSD, 42.7% [95%CI: 38.2-47.1] in UD 10/10, 46.8% [95%CI: 41.1-52.3] in haploidentical) (p=0.013), and the highest NRM (31.2% [95%CI: 23.6-39.2], 18.1% [95%CI: 14.6-21.9] in MSD, 19.7% [95%CI: 16.3-23.3] in UD 10/10, 26.2% [95%CI: 21.6-31.1] in haploidentical) (p=0.001). Of note, LFS at 2 years was reduced for adverse cytogenetic risk (26.1% for adverse, 39.8% for intermediate and 51.9% for favorable risk) (p=0.001) and significantly differed between reduced intensity conditioning (RIC) and MAC (37.4% and 29.8%, respectively) (p=0.033). In multivariate analysis, factors predicting poor LFS were haploidentical donor (hazard ratio [HR] 1.22 [95%CI: 1.01-1.48], p=0.043), adverse cytogenetic risk (HR 1.73 [95%CI: 1.51-1.99], p<0.0001) and time from diagnosis to HSCT (HR 1.03 [95%CI: 1.01-1.06], p=0.009), whereas factors associated with a better LFS were RIC conditioning (HR 0.85 [95%CI: 0.73-0.98], p=0.025) and good Karnofsky performance status (KPS) ≥ 80 (HR 0.74 [95%CI: 0.62-0.88], p=0.0009). Haploidentical transplant correlated with lower OS (HR 1.3 [95%CI: 1.06-1.61], p=0.014) and higher NRM (HR 1.69 [95%CI: 1.18-2.41], p=0.004) and adverse cytogenetic risk was associated with higher RI (HR 1.9 [95%CI: 1.6-2.24], p<0.0001), higher NRM (HR 1.47 [95%CI: 1.15-1.87], p=0.002) and lower OS (HR 1.82 [95%CI: 1.57-2.11], p<0.0001). Moreover, increased age predicted higher NRM (HR 1.31 [95%CI: 1.17-1.47], p<0.0001) and lower OS (HR 1.09 [95%CI: 1.02-1.16], p=0.006). Conclusions Our study identified risk factors for survival outcomes in the challenging setting of allo-HSCT for prAML. The most relevant factors are adverse cytogenetic risk and haploidentical donor, both associated with worse outcomes. Of note, the group of haploidentical transplants presented the highest rate of bone marrow used as a stem cell source, with a possible impact on survival outcomes. Longer time from diagnosis to HSCT negatively impacted LFS, whereas use of RIC and KPS ≥ 80 showed a higher LFS. As concerns secondary endpoints, unrelated donors achieved better OS compared to other donors, and UD 9/10 showed the lowest RI at the expense of the highest NRM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LP-118, a Selective Bcl-2 Inhibitor with Tuned Bcl-Xl Activity, Causes Myeloid Differentiation and Cell Death in Acute Myeloid Leukemia (AML)

Introduction Targeted therapy against bcl-2 has proven efficacious in AML, notably with bcl-2 inhibitor venetoclax. Bcl-2 is one of several proteins in the bcl-2 protein family of anti-apoptotic proteins. Primary and adaptive resistance to venetoclax-induced apoptosis is common and can be due to increased levels of other bcl-2 family proteins besides bcl-2, notably bcl-xL and mcl-1. LP-118, a next generation oral, highly potent, selective bcl-2 inhibitor with tuned bcl-xL activity, has demonstrated preliminary efficacy in venetoclax relapsed/refractory (R/R) acute lymphocytic leukemia cell lines. Methods Cell-based viability assays (MTS) were performed in primary AML cells and AML cell lines. Colony forming unit (CFU) assays were performed on primary AML cells and CD34+ stem cells from healthy donor umbilical cord blood. An in vivo study was conducted in a MOLM-13 AML mouse model (Charles River Laboratories; 8-week-old male NGC mice were injected with AML cells day 1, started treatment day 4). Immunoblot studies were performed to measure on-target activity of LP-118 and bcl-2 family proteins. Flow cytometry and cytospins were performed on primary AML cells and AML cell lines to test for maturation. Results A 24-hour (hr) continuous exposure of LP-118 against AML cell lines demonstrated growth inhibition in all except OCI-AML3 and U937 (also resistant to venetoclax), with an IC50 in the nM range of 2.2 in MOLM-13 (venetoclax IC50 9), 3.5 in MV4-11, 0.75 in HL-60 (venetoclax IC50 2), 86.4 in K562, 7.8 in KG-1 (venetoclax IC50 30), and 4.3 in Kasumi-1 (venetoclax IC50 20). A 72-hr continuous exposure of LP-118 against primary AML cells demonstrated growth inhibition with an IC50 of 12.9uM. Venetoclax R/R primary AML cells were sensitive to LP-118 between 5 and 100uM after 72-hr continuous exposure. Primary AML cells also exhibited a 63% decrease in CFUs at 10uM dose from vehicle, while CD34+ stem cells maintained colony forming capability by CFU assay. Induction of apoptosis was observed with caspase-3 and PARP cleavage on immunoblot in AML cell lines and primary AML cells. Relative baseline levels of bcl-2 family proteins by immunoblot did not correlate with AML cell line sensitivity or resistance to LP-118. Following 4- and 24-hr exposure to LP-118 in MOLM-13, MV4-11, and Kasumi-1 cell lines, BCL-2, BCL-xL, MCL-1, and BCL-W protein levels were not increased relative to vehicle by immunoblot, but BCL2A1 levels increased 3-fold. Similar results were seen following a 24-hour exposure to LP-118 in primary AML cells. Myeloid differentiation was demonstrated in primary AML cells (n=3) and AML cell lines MOLM-13 (n=2) and Kasumi-1 (n=6) by flow cytometric detection of CD11b expression after 48- and 72-hr continuous exposure to LP-118, as well as, morphologically on cytospin images. CD11b was also detected after 48-hr continuous venetoclax exposure on Kasumi-1 cells. Limiting-cell RNA-sequencing is ongoing to identify differentially expressed genes following LP-118-induced myeloid differentiation. In a MOLM-13 mouse model, single-agent LP-118 (50mg/kg PO daily) had a statistically significant median overall survival (mOS) of 24 days compared to control (20 days), but venetoclax (100mg/kg daily for 3 days then weekly) did not (mOS 21 days). There was no significant difference in OS between LP-118+azacitidine (5mg/kg IP days 1-5 of 21-day cycle) and venetoclax+azacitidine. After 60 days, the median OS of LP-118+gilteritinib (15.24 mg/kg PO daily) was not reached and showed increased OS compared to azactidine+gilteritinib and venetoclax+azacitidine. By splenic histopathology, blasts are notably reduced in mice treated with azacitidine+gilteritnib, but spleens are effaced by neoplastic blasts in all other treatment groups. Conclusions LP-118 shows effective cell killing in multiple AML cell lines and primary samples with various molecular and cytogenetic profiles, including a patient sample with a PTPN11 mutation, against which venetoclax is known to be ineffective. Compared to venetoclax, LP-118 is effective at lower IC50 values in AML cell lines and is active in venetoclax R/R primary AML samples. LP-118 also proves efficacious in vivo, exhibiting improved OS and decreased disease burden. Finally, LP-118 induces myeloid differentiation, to our knowledge a novel mechanism of action of bcl-2/bcl-xL inhibition in AML. These data support the recently initiated AML clinical trial (NCT04771572).

A Phase 1, Open-Label, Safety, Tolerability, and Efficacy Study of NC525 in Subjects with Advanced Myeloid Neoplasms

Background With the exception of certain select subtypes, Acute Myeloid Leukemia (AML) remains incurable for a majority of patients with current therapies. Despite recent drug approvals targeting actionable molecular mutations and BCL-2, relapse remains inevitable for most patients with AML due to leukemia-initiating cells and progenitors {i.e., leukemic stem cells (LSCs)} which are resistant to conventional cytotoxic drugs. Novel agents that preferentially target LSCs to lessen or eliminate the risk for relapse while avoiding significant myelosuppression by sparing normal hematopoietic stem cells (HSCs), are highly sought-after anti-leukemia therapeutics. Leukocyte Associated Immunoglobulin-like Receptor 1 (LAIR-1) is an immune inhibitory receptor that is present on most immune cell subsets, including T cells, NK cells, monocytes, macrophages, dendritic cells, and granulocytes. LAIR-1, a surface leukocyte receptor containing two immune receptor tyrosine-based inhibitory motifs (ITIM), binds to endogenous collagen ligands and plays an important role in inducing an immunosuppressive microenvironment. In AML, the engagement of LAIR-1 inhibits intracellular downstream survival signals, leading to decreased GM-CSF-mediated survival and proliferation of AML blasts. LAIR-1 is expressed on AML blasts and LSCs regardless of FAB subtypes, cytogenetics or molecular mutations. Recent work demonstrates that myeloid leukemia cell lines, primary AML blasts, monoblasts and monocytes, and MDS-EB-2 blasts all express LAIR-1. Critically, while high expression of LAIR-1 is characteristic of LSCs, this receptor is minimally detectable on normal HSCs, making it an ideal anti-leukemic target. NC525 is a humanized immunoglobulin gamma 1, kappa (IgG1κ) monoclonal antibody that targets LAIR-1-expressing AML blasts and LSCs. Extensive IND-enabling preclinical studies have shown the following features of NC525: (i) Inhibition of colony formation of AML CD34+ bone marrow cells; (ii) sparing of colony formation and differentiation ability of healthy CD34+ HSCs; (iii) direct ex vivo killing of AML blasts without killing healthy leukocytes; (iv) suppression of AML in both MV4-11 and THP-1 CDX models; (v) restriction of MV4-11 in a humanized murine model, without depletion or suppression of human T cells; (vi) restriction of MV4-11 tumor growth in murine blood, spleen, and bone marrow; and (vii) reduction of AML disease burden in primary patient-derived AML xenograft (PDX) models. Finally, NC525 completely inhibits leukemia propagation in secondary transplant models, suggesting that NC525 is capable of eradicating LSCs. Furthermore, NC525 destroys leukemic cells by ligation-mediated apoptosis of leukemic blasts and stem cells without evidence of an immunomodulatory effect on other immune subsets. Methods This is an open-label, non-randomized, Phase 1 dose escalation study to determine the safety and tolerability of NC525 in adult patients with advanced myeloid neoplasms. This study will also assess the clinical benefit of NC525. Eligible patients must have relapsed or refractory AML, myelodysplastic syndrome (MDS), or chronic myelomonocytic leukemia (CMML) that has progressed after standard therapy. Participants will receive NC525 by intravenous infusion on Days 1 and 15 of each 28-day cycle. During Phase 1, the safety and tolerability of NC525 will be evaluated by exploring escalating dose levels of NC525. In addition, the pharmacologically active dose (PAD), defined as the dose that provides a maximal biologic effect, such as achieving a complete remission (CR), CR with partial hematological recovery (CRh) or CR with incomplete hematological recovery (CRi), and the maximum tolerated dose (MTD) of NC525 will be determined. Safety and tolerability will be further assessed through the expansion of a few dose levels to determine an optimal recommended Phase 2 Dose (RP2D) and administration schedule of NC525, based on the PK/PD profiles. Taken together, preclinical data suggest that targeting LAIR-1 in AML and other myeloid malignancies may be an effective therapy for these diseases with the advantage of specifically eradicating leukemic stem cells and sparing normal HSCs.

Unraveling and Visualizing the Interactions of Acute Myeloid Leukemia with the Bone Marrow Microenvironment in Vitro and In Vivo

The bone marrow (BM) is a dynamic stem cell microenvironment, harboring hematopoietic stem cells (HSCs) and various non-blood components such as endothelium, mural cells, multipotent stromal cells (MSCs) and their derivatives including osteoblasts, adipocytes, and fibroblasts. This stromal niche supports HSC-fate decisions and has been proposed as a major determinant of chemoresistance in hematological malignancies, such as acute myeloid leukemia (AML). Following chemotherapy, AML patients often suffer from relapse, in part due to chemoresistant leukemic stem cells (LSCs). There have been various indications in literature that leukemic cells remodel their niche, creating a permissive microenvironment for LSCs. However, which cell types respond and which underlying molecular mechanisms are involved skewing the compositional changes in the BM microenvironment remain unclear. Here, we aim to investigate how high LSC frequency (LSChigh) versus low LSC frequency (LSClow) burden AML initiates BM niche remodeling in the favor of AML expansion, with a particular focus on the MSC lineages. We further aim to determine which stromal cell types interact with LSCs and how their composition is altered upon AML development. Additionally, we aim to visualize which cell types interact with and are positioned in close proximity to LSCs. We combine single-cell transcriptomics of AML xenograft models and in vitro co-cultures of MSCs with primary AML. First, we set up clinically relevant murine xenograft models in which primary AML cells with either a low vs high LSC frequency (Garg et al., 2019) were injected in immune-deficient NSGW41 mice. As control, we injected cord blood-derived HSPCs. 6-7 weeks after engraftment, we harvested and digested the bones using our established protocol (adapted from Baccin et al., 2020) to recover the stromal cells with high efficiency. Besides, we also kept a femur per mouse for follow-up (multiplexed) immunofluorescence imaging. We enriched for human and mouse HSPCs, human AML cells, and mouse niche cells using FACS-based sorting, followed by scRNA-seq. Additionally, we isolated BM-derived mononuclear cells from healthy donors and AML patients and expanded the MSCs in vitro. We set up 4 independent co-cultures with MSCs and primary AML cells with LSChigh versus LSClow frequency. After 48 hours, we performed scRNA-seq to investigate transcriptomic changes and subsequent variant biological processes in the MSCs. scRNA-seq profiling of the BM niche of our AML xenograft models demonstrates that AML presence significantly impacts the niche composition, leading to the expansion of Cxcl12-abundant-reticular adipocytes (adipo-CAR) and the decline of osteoblasts. When comparing LSChigh versus LSClow, we detect significant differences in abundance of certain stromal cells, including endothelium, adipo-CARs, and fibroblast subtypes. This suggests that the LSC-burden plays a critical role in the extent of niche remodeling. To further uncover the direct effects of LSCs on MSCs, we have established an AML-MSC co-culture system and perform scRNA-seq on the MSCs. In line with our in vivo data, we observe that cultured BM MSCs, especially upon exposure to LSChigh AML, suppress osteoblast lineage genes while overexpressing fibrosis-related genes. We are validating our observations using multiplexed immunofluorescence to further investigate the direct cell-cell communication of LSCs with their microenvironment. Taken together, our data in murine AML models and human AML-MSC co-cultures unravel new insights into the BM stem cell niche composition and highlight a specific LSC-induced skewing of the mesenchymal lineage. Our data helps to better understand to which extent the BM microenvironment impacts AML disease progression and relapse due to residual LSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of Stearoyl-CoA Desaturase Has Anti-Leukemic Properties in Acute Myeloid Leukemia

Metabolic rewiring, a hallmark of acute myeloid leukemia (AML), contributes to the development of resistance to intensive chemotherapy and has been shown to play an essential role in leukemic cell maintenance. Several metabolic alterations have been described in AML, such as dependence on amino acids to fuel oxidative phosphorylation or ability of leukemic cells to switch energy dependence to fatty acid oxidation in response to treatment. However, little is known about the functional significance of the fatty acid synthesis (FAS) pathway in AML. Here we have identified stearoyl-CoA desaturase (SCD), a key enzyme responsible for the conversion of saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA), as a novel therapeutic target in AML. High SCD expression predicts worse survival across several patient datasets, even after correction for gender, age and AML European LeukemiaNet risk category. Moreover, genetic and chemical inhibition of SCD reduces growth and induces cell death in AML cells. Since targeting SCD with currently available inhibitors has been challenging because of their toxicity profile in vivo, we have accessed a novel pharmacological SCD inhibitor with a more favorable toxicity profile, SSI-4 (Modulation Therapeutics Inc., Morgantown, USA). SSI-4 reduces viability in several human and mouse AML models in vitro and in about half of the 36 primary AML samples tested ex vivo. Moreover, SSI-4 displays anti-leukemic effects in AML murine models and patient-derived xenografts (PDX). Since we could not correlate susceptibility to SCD inhibition with any genetic characteristic of AML cells, we aimed to determine the molecular basis of cell sensitivity using phosphoproteomic, proteomic and transcriptomic approaches coupled with metabolic analyses of sensitive and resistant cell lines and primary AML cells. Sensitivity to SCD inhibition directly correlates with rate of de novo FAS, as measured by fatty acid labeling with 13C-glucose. Moreover, modulation of FAS by the AMPK signaling activity dictates sensitivity to SCD inhibition and AMPK activation rescues the anti-leukemic effects of SSI-4. The anti-leukemic effects of SCD inhibition are secondary to the induction of SFA mediated lipotoxicity, as demonstrated by the increased sensitivity of previously resistant cells to SSI-4, when grown in the presence of the SFA palmitate. Conversely, growing cells in the presence of the MUFA oleate, as well as inhibition of FAS by targeting enzymes upstream of SCD, results in resistance to SSI-4, thus confirming that the increased SFA/MUFA ratio drives leukemic cell death. SFA mediated lipotoxicity leads to both increased lipid peroxidation and activation of the unfolded protein response (UPR), namely its PERK branch, which in turn leads to apoptotic cell death. Indeed, inhibition of PERK, but not of other branches of UPR, rescues SCD-mediated cytotoxicity. Despite causing disease debulking in vivo, SSI-4 does not prolong survival in murine AML models as a single agent and the toxic effects of SSI-4 are decreased when AML cells are grown in co-culture with stroma, suggesting a protective role of tumor microenvironment. However, we consistently detect increased lipid peroxidation and DNA damage both in vitro and in vivo following SCD inhibition. We therefore combined SSI-4 with doxorubicin, a known inducer of lipid peroxidation and DNA damage, which is routinely used in AML induction therapy. Indeed, both genetic depletion and pharmacological inhibition of SCD renders AML cells more sensitive to doxorubicin, and addition of SSI-4 to a standard AML chemotherapy protocol consisting of doxorubicin and cytarabine significantly extends survival in AML mouse models. In summary, we identify and validate SCD as a therapeutic target in AML. The novel SCD inhibitor SSI-4 displays anti-leukemic properties both in vitro and in vivo across different AML models and particularly when combined with standard chemotherapy while showing minimal toxicity in vivo. We show that sensitivity to SCD inhibition is driven by metabolic rather than genetic AML features, thus making this therapeutic approach potentially applicable across several AML subtypes, including high risk ones. Finally, the mechanism of toxicity following SCD inhibition suggests that this can be further enhanced by modulating the levels of SFA/MUFA through dietary interventions or by combining it with DNA damaging agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intracellular Calcium Localization Mediates the Activity of Venetoclax in Targeting Acute Myeloid Leukemia Stem Cells

Venetoclax (BCL2 inhibitor) based regimens have significantly improved outcomes for elderly patients with acute myeloid leukemia (AML). While most patients initially respond to venetoclax-based regimens, upfront resistance or relapse on therapy occurs in many. Therefore, elucidating the mechanistic link between BCL2 inhibition and AML cell death is crucial to finding novel therapeutic strategies to combat venetoclax resistance and to discover predictors of venetoclax response. Previous studies have shown BCL2 has a non-canonical function in modulating oxidative phosphorylation (OXPHOS) activity of AML stem cells (LSCs). However, the mechanistic link between BCL2 and OXPHOS has not yet been established. BCL2 has been shown to regulate intracellular calcium localization by modulating the activity of calcium channels at the ER and mitochondria. Optimal mitochondrial calcium concentrations are crucial for cell metabolism as calcium overload or decrease can inhibit OXPHOS activity. In preliminary studies, we established venetoclax increases mitochondrial calcium levels (Fig. 1A) leading to a subsequent decrease in OXPHOS activity and changes in the activity of key calcium dependent TCA cycle enzymes in primary human AML LSCs. Genetic inhibition of BCL2 in LSCs phenocopied the effects of venetoclax treatment. Based on these data, we hypothesized that BCL2 exerts its effect on LSC metabolism by modulating calcium localization between the ER and mitochondria. Furthermore, we sought to explore whether dysregulation of calcium localization by venetoclax is a mechanistic component of drug activity that leads to suppression of OXPHOS. To determine how BCL2 may be influencing mitochondrial calcium levels, we assessed the relationship between BCL2 and SERCA3, a key ion channel responsible for calcium uptake into the ER. Venetoclax disrupted the physical interaction between BCL2 and SERCA3. Mechanistically, venetoclax perturbed this interaction by decreasing SERCA3 protein levels. These findings were recapitulated by genetic inhibition of BCL2 in LSCs. Functional analysis of SERCA3 via siRNA-mediated knock-down in primary AML specimens demonstrated increased mitochondrial calcium levels, decreased OXPHOS activity and decreased engraftment of immune deficient NSG-S mice. Based on these data, we propose a model in which venetoclax disrupts SERCA function leading to less calcium uptake at the ER and more calcium sequestered into the mitochondria leading to overload, decreased OXPHOS activity and decreased LSC function. Next, we evaluated how calcium pathways were impacted in venetoclax resistant AML patient specimens. Intriguingly, venetoclax did not affect mitochondrial calcium levels (Fig. 1B) or the relationship between BCL2 and SERCA3. Therefore, we tested whether inhibiting the activity of MCU, the channel responsible for calcium uptake into the mitochondrial matrix, would target venetoclax resistant LSCs as it was overexpressed in this population. MCU inhibition led to decreased mitochondrial calcium levels, decreased OXPHOS activity and decreased LSC engraftment. These data indicate that venetoclax-resistant AML cells are no longer subject to changes in calcium localization induced by drug treatment but do retain an overall reliance on calcium regulation for LSC function/survival. Finally, we assessed whether our findings could be leveraged to predict venetoclax sensitivity. Analysis of single cell RNA-seq data from venetoclax sensitive versus resistant AML patient specimens (n=19) revealed multiple calcium pathway gene sets were enriched in venetoclax resistant LSCs. Additionally, levels of certain genes including SERCA3 and MCU were differentially expressed in primitive versus monocytic clusters between sensitive and resistant specimens. Taken together, our data demonstrate an unexpected and critical link between BCL2 and calcium signaling in human LSCs. A surprising aspect of our work is that venetoclax, a BH3 mimetic, acts to modulate the interaction with SERCA3. Lastly, our findings suggest that targeting intracellular calcium dynamics may represent a novel therapeutic strategy in the context of venetoclax resistant AML. In addition, we show that there are significant differences in calcium gene signatures between venetoclax sensitive vs resistant AML specimens that may be useful in predicting patient response to venetoclax based regimens. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High MN1 Expression Is Associated with an Lspc-Enriched Phenotype and Glycolysis Representing a New Vulnerability in Acute Myeloid Leukemia

Despite a steady increase in the number of targeted therapies over the past decades, the 5-year overall survival (OS) rate remains poor in acute myeloid leukemia (AML). Currently, high expression of the oncogenic Meningioma 1 (MN1) is associated with poor OS in AMLs with normal karyotype, and we correlated its expression with a more immature cellular composition in AML samples. Hence, we hypothesized that supplementation of AML cells that usually fail to engraft in xenograft models, such as acute promyelocytic leukemia (APL), with MN1 would promote in vivo expansion in mice allowing the study of potential therapeutic vulnerabilities. The in vitro experiments were performed in human AML cell lines, primary AML cells (n=39), and isolated healthy CD34+ cells. MN1-overexpression (OE) was lentivirally performed. Bioenergetic profiles were evaluated by a Seahorse analyzer. Gene expression was measured by RNA-sequencing/qPCR. Protein levels were measured by immunofluorescence microscopy. Viability and surface protein expression was evaluated by flow cytometry. For in vivo studies, eight-weeks old female NSGS mice were transplanted with MN1-OE primary APL blast cells, and mice were followed for 12 weeks. Cells from the mouse organs were isolated, analyzed, and sorted for human APL blasts (CD45+CD117+CD33+) for ex vivo studies. We show that genetic MN1-OE in APL cells resulted in increased cell survival upon cytotoxic therapy, and decreased sensitivity to ATRA-induced terminal differentiation in short and long term cultures (3/8 days). In healthy CD34+ cells, we observed that MN1-OE was able to increase cell proliferation and stemness properties in CMP-sorted (CD34+CD38+CD123+CD45RA-) but not in GMP-sorted cells (CD34+CD38+CD123+CD45RA+), showing in human models the results from Heuser et al., 2011 in murine MN1-model. Co-expression of MN1+PML-RARα in healthy CD34+ cells resulted in superior expansion with retention of the CD34 marker, in comparison with the single hits. At the molecular level, APL cells with MN1high displayed transcriptional programs associated with "HYPOXIA"/"HSC-UP", whereas MN1low levels correlated with "OXPHOS-UP"/"GMP-UP". Despite no evident differences in Seahorse analysis, MN1-OE cells exhibited increased levels of the glycolytic enzyme PDK1. Functionally, MN1-OE cells were more sensitive to glycolytic inhibitors 2,2-dichloroacetophenone (a PDK1 inhibitor) and 2-deoxyglucose, revealing glycolysis as a metabolic vulnerability in MN1high AMLs, which was further validated in primary AML samples. These data are in line with our previous report indicating that more immature AML subtypes typically display a more glycolytic metabolic profile (Erdem et al, 2022). MN1 is an intrinsically disordered protein containing a long polyQ-stretch that is required for transformation (Riedel et al, 2021). We questioned whether the expression of chaperone proteins would be necessary for the appropriate functioning of MN1. We observed that expression of the DNAJB6 isoform 2 (DNAJB6b) correlated with MN1 expression across a large panel of genetic subtypes of AML, including more immature AML subtypes, such as the TP53-mutant AML but also in APL. shRNA-induced knockdown in AML cells of DNAJB6b resulted in cytosolic retention of MN1, associated with reduced cell survival. In vivo, the patient-derived xenograft model showed engraftment of APL samples upon MN1-OE, with the colonization of secondary organs and splenomegaly, which was not observed in the control. Flow cytometry analysis revealed that MN1-engrafted mice displayed increased levels of APL blasts in comparison with control, which had a large proportion of mature myeloid cells (CD33+CD11b+). Ex vivo screen of engrafted cells revealed increased resistance to ATRA-induced differentiation in MN1-OE sorted blasts. We provide further evidence to support the role of MN1 as an oncogenic hit allowing in vivo studies of difficult-to-engraft AML subtypes. Functionally, high MN1 expression resulted in decreased differentiation, retention of stem-like properties, and metabolic shift towards a more glycolytic state. We demonstrated that DNAJB6b controls the cellular localization of MN1, with its downregulation associated with the loss of MN1 supportive functions. Finally, our findings suggest that targeting glycolysis can be leveraged as a therapeutically actionable mechanism for driving cell death in MN1high AMLs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

FLT3-ITD Degrades CEBPA Via the Ubiquitin-Proteosome Pathway

FLT3 internal-tandem-duplication (FLT3-ITD) mutations are highly recurrent in acute myeloid leukemias (AML). Although secondary messenger impacts of these receptor tyrosine kinase mutations are well characterized, downstream mechanisms by which myeloid precursors are transformed remain unclear. One clue is that FLT3-ITD and CEBPA bi-allelic mutations are mutually exclusive, suggesting that FLT3-ITD might impact CEBPA, a master transcription factor mandatory for granulo-monocytic lineage-differentiation. In fact, FLT3-signaling has been shown to phosphorylate CEBPA at serine 21 (S21-P), and inhibition of this post-translational modification resumed granulocytic-differentiation of FLT3-ITD AML cells (Radomska et al, J Exp Med 2006). How S21-P post-translational modification of CEBPA blocks granulocyte lineage-differentiation, however, was unknown. To examine FLT3-ITD impacts on CEBPA function in primary AML cells, we examined expression of CEBPA target genes (24 genes identified by CEBPA ChIP-seq, Jakobsen et al, Genome Res 2013): CEBPA target genes positively correlated with CEBPA mRNA expression in normal granulopoiesis and in primary FLT3/CEBPA-wildtype (WT) AML cells (n=112) (Pearson correlation coefficient [ρ] 0.36, p<0.0001), but not in primary FLT3-ITD AML cells (n=35)(ρ =-0.15, p=0.4). Moreover, CEBPA target genes were almost 2-fold less expressed in the FLT3-ITD AML cells (p<0.0009), despite CEBPA mRNA expression comparable to the WT AML cells (RNA-sequencing, TCGA). To investigate the disconnect between CEBPA mRNA expression and CEBPA target gene activation, we analyzed CEBPA protein, and found very low amounts in FLT3-ITD AML cells MOLM13 and MV411 compared to large CEBPA protein amounts detected in WT THP1 AML cells, both by Western blot and immuno-fluorescence, again despite similar CEBPA mRNA expression. This observation complemented published data from others that FLT3-ITD introduction into murine 32D cells suppressed total Cebpa protein (Mizuki et al, Blood 2003; Zheng et al, Blood 2004). The FLT3-inhibitors sunitinib and gilteritinib decreased CEBPA S21-P, increased CEBPA total protein measured by Western blot and immunofluorescence, and induced terminal granulocytic differentiation in the FLT3-ITD MOLM13 and MV4-11 AML cells, but not FLT3-WT THP1 or OCI-AML3 cells. Moreover, the proteosome inhibitors bortezomib or MG132 increased S21-P-, ubiquitinated-, and total-CEBPA protein in the FLT3-ITD but not the FLT3-WT AML cells. We therefore further investigated interactions of endogenous CEBPA with the ubiquitin-proteosome system (UPS): unbiased analyses by immunoprecipitation (IP)-LCMS/MS demonstrated 3-4 fold enrichment of the deubiquitinase USP7 and E3 ubiquitin ligase UHRF1 in the CEBPA interactome of FLT3-ITD versus FLT3-WT AML cells. Reverse analyses of the endogenous USP7 protein interactome by IP-LCMS/MS and IP-Western blot confirmed USP7 interactions with CEBPA and UHRF1 and demonstrated USP7 phosphorylation at serine-18 (S18-P), a candidate for regulation by FLT3. The small molecule P22077 alters and inhibits USP7 catalytic site conformation: P22077 shifted USP7 deubiquitinase interactions from UHRF1 to CEBPA, decreased UHRF1 and increased total CEBPA (Figure). FLT3-ITD AML cells were >2-fold more sensitive to P22077 than FLT3-wt AML cells (IC50 5.8 and 7.1 µM for MV411 and MOLM13 versus 13.65 and 21.7 µM for THP1 and K562 cells) (Figure). Another mystery in FLT3-ITD AMLs is relative chemo-resistance even though p53 is wild-type - USP7 regulates p53, and thus FLT3-ITD regulation of USP7 may contribute to chemo-resistance, a link we are currently consolidating. In sum, FLT3-ITD signals for CEBPA protein degradation via USP7 and UHRF1, to thereby decouple proliferation from onward lineage-maturation in committed granulo-monocytic progenitors. Overall, this pathway explains FLT3-ITD: (i) origin in committed granulo-monocytic progenitors; (ii) link to suppressed CEBPA-target genes; (iii) mutual exclusivity with bi-allelic CEBPA mutations; (iv) differentiation-syndrome upon treatment with FLT3-inhibitors; (v) sensitivity to UPS inhibitors; (vi) mutual exclusivity with TP53-mutations yet chemo-resistance, and can inform understanding of how other receptor tyrosine kinase mutations, e.g., KIT mutations, transform committed lineage-progenitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax Plus Hypomethylating Agent (HMA) Based Induction for Treatment-Naive Older/Unfit Patients with FLT3-Mutated Acute Myeloid Leukemia (AML)

Introduction: FMS-like tyrosine kinase 3 (FLT3) mutation (m) is one of the most prevalent mutations in acute myeloid leukemia (AML), and it is associated with an increased risk of relapse and decreased overall survival (OS). The standard of care therapy for physically fit treatment naïve patients is intensive chemotherapy with FLT3 inhibitor (i) (midostaurin). For older/unfit patients, venetoclax-based induction is preferred as per NCCN guidelines. We explored the clinical outcome of FLT3m AML patients who were ineligible for intensive chemotherapy and treated with a hypomethylating agent (HMA) plus venetoclax (Ven) based induction at Mayo Clinic. Methods: Thirty FLT3m AML patients who were ineligible for intensive chemotherapy and received HMA+Ven induction at Mayo Clinic between June 2019 and May 2022 were analyzed for clinical characteristics and treatment outcomes. Patients who received HMA+Ven for the relapsed or refractory disease were excluded. All patients received at least one cycle of HMA, either azacitidine (75 mg/m2 from day 1 to 7) or decitabine (20 mg/m2 from day 1 to 5). Responses were assessed according to the 2022 European Leukemia Net (ELN) criteria. Results: The baseline characteristics are summarized in Table 1. Most of the patients (n=27, 90%) were ≥ 65 years old with a median age of 74 years (range, 59 to 88). Majority of the patients (n=16, 53%) had de novo AML, 8 (27%) patients had secondary AML, 4 patients (13%) had primary AML with MDS-related changes, and 2 patients (7%) were diagnosed with therapy-related AML. Two-thirds of the patients (n=20, 67%) had diploid cytogenetics (CG) and 10 (33%) patients had additional cytogenetic (CG) abnormality at diagnosis. Twenty-six (87%) and 4 (13%) patients had FLT3 ITD and TKD mutation subtypes, respectively. The median baseline FLT3m allelic ratio was 0.39 (range, 0.1-0.7). Most frequent reported mutations (≥ 5%) were DNMT3A (40%), NPM1 (37%), spliceosome mutations (SF3B1, U2AF1, and SRSF2; 30%), RUNX1 (23%), and TET2 (17%). Seventy percent of the patients achieved complete remission with or without count recovery (CR/CRi) (n=21), 13% (n=4) of the patients had partial remission, and 17% (n=5) of the patients had refractory disease to HMA+Ven induction. The median time to achieve CR/CRi was 35 days (range, 22-230). Most CR/CRi patients (86%, n=18) continued consolidation therapy with HMA+Ven. Until changing the HMA+Ven regimen for any reason, the median number of HMA+Ven cycles received was 6 (range, 1-13). Only one patient received allogeneic stem cell transplant allo-HSCT after achieving remission and received gilteritinib maintenance therapy post-transplant. Six (29%) of the CR/CRi patients had relapse with a median time to relapse of 6 months (range, 1-10). The median duration of response was 13 months (95% CI: 6.71-19.28) (Figure 1A), and the median OS was 15 months (95% CI: 8.89-21.10) (Figure-1B). FLT3i (gilteritinib) was received in 6 patients (20%) post HMA+Ven; 2/6 received as maintenance therapy and 4/6 for relapse disease. In the univariate analysis for OS, diploid cytogenetics (p<0.0001) and non-high risk cytogenetics (p=0.04) were associated with significantly better OS (Figure-1D/E). AML sub-type (de novo vs sAML) (p=0.97), NPM1 mutation at diagnosis (p=0.39), FLT3m subtype (ITD vs TKD) (p=0.79) or allelic ratio at diagnosis (<0.5 vs >0.5) (p=0.89), and HMA sub-type (azacitidine vs decitabine) (p=0.43) were not significantly associated with difference in OS (Figure-1C/F/G/H, respectively). Conclusions: In summary, HMA+ Ven showed significant activity in treatment naïve FLT3m AML patients who were ineligible for intensive chemotherapy. HMA+Ven can be used as a backbone in combination with FLT3 inhibitor to improve outcome further and for successful transition to curative intent therapy such as allo-HSCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Real-World Outcomes and Cardiac Implications of FLT3 Inhibitors in Acute Myeloid Leukemia

Background:FLT3 is a member of the class III receptor tyrosine kinase family and constitutively activating mutations of FLT3 are found in ∼30% of adult patients with acute myeloid leukemia (AML). Clinically, FLT3-ITD mutations are associated with earlier time to relapse and poorer overall survival (OS). FLT3 inhibitors are divided into first generation multi-kinase inhibitors (such as sorafenib, lestaurtinib, midostaurin) and next generation inhibitors (such as quizartinib, crenolanib, gilteritinib) or based on their specificity of FLT3 inhibition [type I inhibitors (midostaurin, gilteritinib) inhibit FLT3 ITD and TKD mutations whereas type II inhibitors (sorafenib, quizartinib) have only FLT3 ITD activity]. Though FLT3 inhibition has showed marginal survival benefit in the front line setting for AML, some of the benefits may be offset by cardiovascular toxicities such as cardiomyopathies, QT prolongation and arrythmias. Therefore, outcomes and cardiac safety profile of FLT3 inhibitors in AML at our institution was investigated in the real-life scenario. Methods: In this retrospective study, we included all FLT3MTAML patient with ECOG status of ≤ 2 at our institute who either received FLT3 inhibitors (1) at diagnosis, (2) in a study after being refractory to ≥1 treatment, or (3) relapsing after achieving prior remission. We analyzed the safety and tolerability of type I and type II FLT3 inhibitors in terms of dose-limiting toxicities (DLT) and treatment-emergent adverse events (TEAE) from a cardiac standpoint. Baseline 2D Echocardiograms, EKGs pre- and post-treatment, metabolic profile through each hospitalization, major cardiac events in correlation to prior cardiac history and electrolyte abnormalities were included in the final analysis. Results: From June 2016 to April 2021, 63 FLT3MT AML patients were treated at our institution, 41 (65%) with primary AML and 22 with secondary AML (35%). 52 patients (83%) had ITD and 11 (17%) had FLT3 TKD mutations. Furthermore, 29 patients (56%) with FLT3 ITD and 3 (27%) with FLT3 TKD were treated with FLT3 inhibitors. Of the patients who received FLT3 inhibitors, 21 (66%) received type-2, and 11 (34%) got type I FLT3 inhibitors. 17 patients received FLT3 inhibitors as mono-therapy, and 15 in combination with chemotherapy. Median age of patients on FLT3 inhibitor treatment was 77 years (range 40- 83), and median duration of treatment was 96 days (range 45-180). CRi and CRp were seen as the best response in 12 (38%) and 5 (17%) of the 32 FLT3MT patients respectively, and the median time to CRi/CRp was 33 days (range 22 to 36). Time to relapse in patients with CRi/CRp was 4.5 months (range 2 to 7.5 months). Median overall survival of FLT3 ITD patients treated with type II inhibitors was 18 months, versus 14.5 months for type I inhibitors (p = 0.782), and 5.6 months in patients who did not receive FLT3 inhibitor therapy. The most common non-cardiac treatment emergent adverse events (TEAEs) in patients on FLT3 inhibitors were fatigue, muscle weakness, anemia, and decreased appetite. In the total FLT3MT AML cohort, cardiac adverse events were observed in 16 patients (16/63), 12 of which were on FLT3 inhibitors and 4 were not on FLT3 inhibitors (p = 0.0006). All cardiac TEAEs with FLT3 inhibitor therapy were observed only with type II inhibitors (p=0.0018). Notably, 12 (12/32) patients were classified to have serious cardiac TEAEs, and all of them were receiving type II FLT3-inhibitors. Serious cardiac TEAEs included Torsades in 5 (41%) patients, recurrent ventricular fibrillation in 2 (17%), third-degree AV block necessitating emergent pacemaker in 2 (17%), permanent atrial fibrillation in 2 (17%), and asystole culminating in death in 1 patient (8%) [Fig.1]. 12 patients who developed cardiac TEAEs with FLT3 inhibitors had hyperkalemia, 5 had a preceding history of atrial fibrillation and PVC, suggesting a decreased threshold of development of cardiac TEAEs in the presence of known risk factors. Conclusions: Type II FLT3 TKIs had worse cardiac safety profile in our cohort and an increased risk of cardiac toxicity, particularly in patients with prior reduced ejection fraction or A-fib. A careful cardiac history and management of predisposing conditions to serious cardiac events could help improve drug tolerance, morbidity, and mortality. Further studies are required to elucidate the precise mechanisms of increased incidence of cardiac arrythmias and ways to mitigate them. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DIACC2010, Sole-in-Class Selective Inhibitor of Kinesin KIF20A, Has Potent Preclinical Efficacy in Acute Myeloid Leukemia

Context: Despite recent therapeutic advances in Acute Myeloid Leukemia (AML) and the decent proportion of complete remission after induction chemotherapy, relapse rate is still high and long-term survival does not exceed 20%. The therapeutic arsenal of AML could benefit from differentiated strategies. Mitotic kinesins are essential regulators of cancer cell replication and migration. The microtubule associated motor protein KIF20A (also called MKlp2), a member of the kinesin-6 family, plays an essential role during cytokinesis and is also involved in the fission of RAB6-positive vesicles from Golgi membrane, therefore contributing to intracellular vesicular trafficking. KIF20A plays a critical role in the development and progression of many cancers, and its high expression is associated with disease progression and poor survival outcome. More specifically, immature hematopoietic cells, exhibit high KIF20A expression, whereas mature peripheral blood cells do not. Herein we describe the preclinical anti-leukemic efficacy of DIACC2010, sole-in-class selective KIF20A inhibitor. Methods: We used cell lines (U-937, KG-1, CEM, KASUMI, SKM-1, MOLM-14, HL-60, MV-4-11, THP1) provided by ATCC and a Cell line-Derived Xenograft models to demonstrate the anti-leukemic activity od DIAC2010. Briefly, NOD.Cg-Prkdc scid/J (NSG) mice were obtained from Charles River France and maintained under specific pathogen-free conditions. Animals were injected via tail vein with 0.2 106 viable human leukemia cells. Mice were treated with 0.9% NaCl or Diacc2010 (200 mg/kg IV; once a week). Daily monitoring of mice for symptoms of disease determined the time of killing for animals with signs of distress. In addition, primary AML samples were obtained after informed consent. Cells were transplanted to NSG mice, collected after the 3rd transplantation and drug tests were performed ex vivo. All animal experiments were performed in compliance with the laws and in agreement with the French Guidelines for animal handling and approved by animal ethics committees (Agreement no. APAFIS#6743). Golgi imaging was performed using anti-GM130 antibody. Cell cycle, Caspase-Glo 3/7 and Annexin V/PI tests were performed according to the manufacturer's protocols. Results: DIACC2010 demonstrated potent and consistent cytotoxic activity in vitro against a panel of 9 human AML cell lines, with median IC50 of 40 nM (range 10-77 nM). In the same experimental conditions, cytarabine (CYTA) had median IC50 of 207 nM (range 4-1580 nM). DIACC2010 was subsequently evaluated in vivo in xenograft models of AML cell lines. DIACC2010 significantly improved overall survival as compared to CYTA in all models, with dose-dependent efficacy as shown in Figure A ( MOLM14 cells. Next, DIACC2010 cytotoxic activity was evaluated in vitro on 8 primary AML patient samples, IC50 ranged from 57 nM to 728 nM across the tested samples. DIACC2010 antitumoral activity is currently evaluated in Patients Derived Xenograft model. In parallel, no in vitro toxicity of DIACC2010 was observed on human normal cells such as peripheral blood mononuclear cells (PBMC) and primary hepatocytes (IC50 >50 µM). On a mechanistic standpoint, DIACC2010-exposed AML cells displayed characteristic Golgi scattering, observed within 1 hour of treatment and majoring at 6 hours confirming target engagement (Figure B). In parallel, we observed a strong block in cell cycle in G2/M after 6 hours of treatment (45% of cells versus 21% in the control condition). This was associated with a high caspase activation that occurred mainly after 24 hours of treatment. This cascade led to cell death assessed by annexin V/PI between 24 and 48 hours after treatment initiation. Nevertheless, caspase inhibitors ZVAD-fmk and Emricasan partially rescued DIACC2010 cell death, although these inhibitors significantly rescued caspase activation induced by Venetoclax, a BCL-2 inhibitor triggering intrinsic apoptosis commonly used in AML treatment. We ruled out necroptosis and ferroptosis as cell death pathways triggered by DIACC2010 using pharmacological inhibitors. These data strongly suggests that DIACC2010 induces a non-redundant cell death pathway that could be advantageously used to kill resistant AML cells. Conclusion: Altogether, these results confirm the relevance of KIF20A-directed therapeutic approaches and support the development of DIACC2010 for the treatment of AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ectopic Viral Integration Site-1 Promotes NRAS/ERK Pathway through Epigenetic Silencing of microRNA-124 in AML

BACKGROUNDː Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by genetic mutations that promote proliferation of myeloid progenitors and prevent their differentiation. Over-expression of Ectopic Viral Integration site-1(EVI-1) is related to the poor prognosis in myeloid leukemia, but the underlying mechanism remains unclear. METHODSː Using qRT-PCR and western blotting, we quantified expressions of EVI-1, NRAS and ERK/p-ERK in leukemia cell lines and PBMCs. Using WTS-8 and cell cycle analysis, we further investigated whether downregulation of EVI-1 by siRNA can inhibit cell proliferation. Microscopic observation of peripheral blood cells from EVI-1 transgenic zebrafish and WT control were analyzed by Wright Giemsa staining. Using miR-seq, qPCR, dual-luciferase reporter and coimmunoprecipitation assays, we revealed the relationship between EVI-1, miR-124 and NRAS. RESULTSː EVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). In a transgenic zebrafish model, EVI-1 mediated higher mortality and induced immature hematopoietic cells in the blood circulation, suggesting its oncogenic role. Furthermore, our results suggested that EVI-1 upregulated NRAS expression, thereby activating the RAS/ERK pathway through epigenetic silencing of a potent NRAS suppressor, miR-124. In this study, we found that EVI1 physically interacts with Dnmt3a to form a protein complex that targets and binds to regulatory elements of miR-124. CONCLUSIONSː Overall, the current findings demonstrate that EVI-1 overexpression converges on the regulation of miR-124 promoter methylation and activation of the RAS/ERK pathway in AML carcinogenesis, and suggest EVI-1 and/or miR-124 as therapeutic targets for this dismal disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Translatome Proteomics Identifies Autophagy As a Resistance Mechanism to on-Target FLT3 Inhibitors in Acute Myeloid Leukemia

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 are among the most frequent mutational targets in acute myeloid leukemia (AML), drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) such as quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and often occurs in the absence of identifiable genetic causes, suggesting that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. We therefore systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD+ AML using recently developed translatome proteomics to measure changes in the nascent proteome in combination with kinase signaling mapping by phosphoproteomics to identify therapy resistance mechanisms to FLT3 inhibitors in vitro. This systems-biology approach showed that the autophagy network escapes global translation repression caused by FLT3i and pinpointed autophagy induction mediated by the AKT-mTORC1-ULK1 axis as the most dominant stress response mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD+ cells in vitro and in primary human AML cells ex vivo. Inhibition of FLT3-ITD signaling led to decreased phosphorylation at AKT S473, decreased phosphorylation at the AKT substrate mTOR S2448, and decreased phosphorylation at the mTORC1 substrate ULK1 S757. CRISPR/Cas9-targeting of TSC2, a negative mTORC1 regulator, ULK1 or ATG3, a core autophagy protein required for autophagosome formation, significantly attenuated or largely abolished autophagy induction by FLT3i. Pharmacological autophagy inhibition (ROC-325, Lys05) or genetic ablation of autophagy (shRNA-mediated knockdown of ATG3, ULK1 or its target Beclin1) increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In immunodeficient mice xenografted with human FLT3-ITD+ AML cells, oral co-treatment with gilteritinib and autophagy inhibitor ROC-325 synergistically impaired leukemia progression and extended overall survival. Our findings show the utility of functional translatome proteomics to systematically investigate cell-autonomous therapy resistance mechanisms, identify a molecular mechanism responsible for primary on-target FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

Comparison of Data from Fresh and Frozen AML Samples for Functional Drug Testing

Functional testing on cancer patient samples is common in cancer research, but the methodology and type of samples used can vary considerably between studies. In 25 recent studies, where functional testing was performed on blood cancers, half used fresh samples, while others included only frozen or both sample types. Here, we investigated how sample handling influences functional testing in primary acute myeloid leukemia (AML) patient samples. To look for systematic changes in cell population induced by sample handling, we first compared cellular function between frozen (n=70) and freshly isolated (n=112) bone marrow mononuclear cell samples from patients with AML using a high-throughput functional drug testing assay with 528 clinically applied and emerging drugs. We then specifically collected paired samples (n=10) before and after cryopreservation using drug testing and flow cytometry as readouts, including a flow cytometry-based drug testing assay for selected compounds.The Z'-factor, an assay quality indicator, was similar between frozen and fresh sample groups. However, overall cell viability and growth rate were significantly lower in the frozen samples. Interestingly, in fresh samples, delays from sampling to the start of the assay had no effect up to 72h. When comparing overall drug efficacies, most of the drugs tested correlated highly between sample types, but 29 out of 528 drugs showed a notable systematic difference in drug sensitivity. Samples that had been frozen were more sensitive to drugs such as kinase inhibitors (amcasertib, dinaciclib), as well as apoptotic modulators (S-63845), and proteasome inhibitors (VLX1570). Additionally, fresh samples were more sensitive to paclitaxel than frozen ones across the paired samples.The cell composition of paired samples was also affected by cryopreservation, with a reduction in the frequency of cells expressing c-KIT (CD117) and a reduction of cells with high granularity (side scatter). Similar effects could be observed for the flow cytometry-based assay where frozen samples showed a cryopreservation-potentiated reduction in CD34+,CD11b+ and CD56+ cells after proteasome inhibitor treatment, and a reduction in CD56+ cells after taxane treatment. In conclusion, drug responses are in general highly correlated between fresh and frozen samples for about 95% of the drugs tested. However, careful consideration should be given to the specific drugs and cell populations of interest before deciding on sample handling methodology. This may be of particular importance when investigating specific drugs and phenotypes affecting cell proliferation and maturation stage, as sample handling may induce systematic differences and confound interpretation.

A Phase 1 Study of WU-NK-101 in Patients with Relapsed or Refractory (R/R) Acute Myeloid Leukemia (AML)

Background: Natural Killer (NK) cells are central to anti-tumor immunity, with the capacity to directly kill tumor cells by recognizing markers of cellular stress or abnormality. Through cytokine reprogramming, NK cells can gain memory-like features that augment their anti-tumor potential. WU-NK-101 is a non-engineered, cytokine-reprogrammed, expanded, and cryopreserved off-the-shelf NK cell product derived from peripheral blood mononuclear cells. WU-NK-101 demonstrates many features synonymous with memory NK cells, such as enhanced activity, proliferation, and persistence without the requirement of additional engineering strategies, overcoming limitations associated with conventional NK cell therapies. In addition, WU-NK-101 demonstrates potent in vitro and in vivo cytotoxicity against tumor cells, and an enhanced and adaptive metabolic fitness contributing to resilience within an adverse and immunosuppressive TME. Through large-scale feeder-cell-free expansion, WU-NK-101 enables multi-dose treatment of several patients from a single apheresis product (Mathyer M et al. SITC 2021) Multi-dosing may further augment anti-tumor activity leading to enhanced patient benefit without a substantial increase in patient risk. Methods: This is a Phase 1, open-label, dose escalation, and cohort expansion study (NCT05470140) designed to characterize the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity, and preliminary anti-leukemic activity of WU-NK-101 in R/R AML. In the Dose Escalation Phase, up to 18 patients will be treated in up to 3 dose levels (DL), starting with 300 x 106 for DL 1, 900 x 106 for DL2 and 1800 x 106 for DL3, with 1 potential de-escalation DL. Successive cohorts of 3 to 6 patients will be enrolled using a standard 3 + 3 design. Once the maximum tolerated/administered dose is defined, 6 additional patients will be enrolled in the Cohort Expansion Phase to further characterize the safety, tolerability, as well as determine the recommended Phase 2 dose of WU-NK-101. Patients will receive a lymphodepleting chemotherapy regimen (cyclophosphamide 50mg/kg and fludarabine 125mg/kg) starting on days -6 to -2 prior to receiving WU-NK-101. WU-NK-101 will be administered on Days 1, 8, and 15 in a 28-day cycle. All patients will be hospitalized for a minimum of 3 days following the first infusion of WU-NK-101 and will receive with recombinant human interleukin-2 every other day of the cycle. Treatment will be limited to 1 cycle in dose escalation. Patients achieving partial or complete remission in cohort expansion may receive re-induction and/or consolidation, for a total of up to 4 cycles. Eligible patients must have a confirmed diagnosis of primary or secondary AML (any subtype except acute promyelocytic leukemia) according to World Health Organization 2016 classification and be unlikely to benefit from standard of care chemotherapy. Eastern Cooperative Oncology Group Performance Status must be ≤ 2 at screening. Patients with known central nervous system involvement are eligible if they have been treated and cerebrospinal fluid is clear for at least 2 weeks prior to enrollment. Patients with extramedullary disease are permitted if bone marrow blast count is >5%. Key exclusions are circulating blast count >30,000/µL by morphology or flow cytometry (cytoreductive therapies such as leukapheresis or hydroxyurea are allowed); uncontrolled or untreated bacterial or viral infections; uncontrolled angina, severe uncontrolled ventricular arrhythmias, or ECG suggestive of acute ischemia or active conduction system abnormalities; and severe renal impairment. Response assessment will take place on Day 28 (+/-3 days), by bone marrow aspirate and core biopsy, and response will be defined per 2022 European LeukemiaNet criteria. Response rate, duration of response, and mortality rate at 1 and 3 months will be evaluated.

Relocalization of PU.1 Underlies the On- and Off-Tumor Effects of SWI/SNF Blockade in AML

SWI/SNF ATP-dependent chromatin remodeling activity has been identified as a strong dependency of acute myeloid leukemia (AML). In AML cell lines, genetic knockdown or chemical inhibition of the SWI/SNF ATPases SMARCA4 (BRG1) or SMARCA2 (BRM) by the selective small-molecule inhibitor BRM014 reduces proliferation and induces leukemic differentiation. These effects arise because SWI/SNF inhibition reduces DNA accessibility at binding sites of PU.1, a factor essential for driving high levels of MYC in leukemic cells. However, given that both SWI/SNF and PU.1 are essential in hematopoietic development, important questions regarding the use of SWI/SNF inhibitors against AML remain. First, the chromatin features that influence SWI/SNF-dependent PU.1 regulation within healthy hematologic and leukemic cells have not been established. Furthermore, the ability of SWI/SNF inhibitors to target leukemic cells within the bone marrow has remained uncertain, and the effects of these drugs in an immunocompetent setting have not been reported, despite the essential functions of SWI/SNF and PU.1 in normal hematopoiesis. Finally, the sensitivity of primary human AML samples and normal hematopoietic cells has also not been established. As a result, many questions regarding the nature of the therapeutic window for SWI/SNF inhibition against AML have remained unresolved. Here we evaluate SWI/SNF inhibition using BRM014 in clinically relevant models and examine the consequences of the deregulation of SWI/SNF-dependent PU.1 sites. We demonstrate that inhibition of SWI/SNF shifts PU.1 occupancy from enhancers to promoters in both leukemic and healthy hematopoietic cells. In AML, the shift of PU.1 away from enhancers to the promoters of differentiation-related genes results in loss of MYC expression and differentiation of leukemic cells. In non-leukemic mice, SWI/SNF inhibition suppresses PU.1-dependent B cell development and bone marrow-derived monocyte populations, reflecting off-tumor effects caused by SWI/SNF-dependent PU.1 relocalization. These effects influence peripheral immune cells, as well lineage-committed hematopoietic progenitors within the bone marrow. Moreover, we observe profound therapeutic response to SWI/SNF inhibition in an immunocompetent AML mouse model (Figure 1). In vivo, BRM014 induces differentiation of peripheral leukemic blasts and reduces the leukemic stem cell burden in bone marrow but also induces lymphocytopenia due to its off-tumor effects on PU.1. Nevertheless, the robust regression of leukemic burden seen in vivo over a short two-week treatment period provides a compelling justification for continued study of SWI/SNF inhibitors in the treatment of AML. Further, response of primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and AML specimens highlight that normal hematopoiesis is affected at concentrations similar to those needed for therapeutic response in AML (Figure 2). Hence, hematopoietic and immune-related side effects, particularly at higher doses and over the duration of treatment, will be important considerations for clinical use of SWI/SNF inhibitors against AML and other tumors. Overall, our results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comprehensive Age-Stratified Impact of NPM1 Mutation in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) with NPM1 mutation (MT) in the absence of FLT3-ITD is a favorable prognostic risk group in various stratification schemes, including the recently published machine learning-based, clinical-feature agnostic analysis of AML mutations (Awada et al., Blood 2021). However, prior large clinical trials based large registry data (Ostronoff et al., JCO 2015) observed that the positive prognostic impact of NPM1MT is not general as it does not seem to extend to elderly patients above 65 ys. in contrast to prior smaller studies. In this group, a formal, extensive analysis has not been performed on patients treated outside clinical trials. AML cohort from Karmanos Cancer Institute (n=52) was enhanced by a publicly available metanalytic cohort consisting of various sub-studies (Awada et al., Blood 2021) to a total of 2,165 AML patients to identify and validate the age-stratified analyses of the impact of NPM1MT in AML. Only patients whose NPM1 mutation status was available were included in the study. Baseline clinical and molecular characteristics were noted. Chi-square test was used to study various parameters as described, and Kaplan Meir curves were used for survival analyses. In total, 25% (533/2,165) of the patients carried NPM1MT. Male: Female ratio was 0.92. NPM1MT was enriched in primary (p) AML compared to NPM1WT [94 vs. 74%, p=0.0001], whereas there were less secondary (s) AML in NPM1MT compared to NPM1WT [6 vs. 26%, p=0.0001]. NPM1MT was less associated with abnormal karyotype versus NPM1WT [16 vs. 63%, p<0.0001]. Median variant allele frequency (VAF) in evaluable patients was 38% [range, 5-60%], and all mutations were canonical frameshift. Median overall survival (OS) was significantly better in NPM1MT compared to NPM1WT patients [24 vs. 17.5 mo., p=0.003]. When grouped by FLT3-ITD mutational status, NPM1MT patients had a better OS compared to NPM1WT in FLT3-ITDNEG group [29 vs. 17 mo., p=0.00045]. Whereas there was no difference in OS in the FLT3-ITDPOS group [15 vs. 21 mo., p=0.461]. NPM1MT patients with pAML had a better OS compared to NPM1MT patients with sAML [31 vs 9.6 mo., p=0.001]. When stratified by age groups, NPM1MT/FLT3-ITDNEG patients had a significantly higher OS than NPM1WT/FLT3-ITDNEG patients in the 55-65 year age group (75 vs. 20 mo., p=0.002). After splitting this group, the OS benefit was preserved in the 55-60 (58.5 vs. 16 mo., p=0.041) and 60-65 year group (mOS not estimable vs. 21 mo., p =0.011). There was no difference in OS for NPM1MT/FLT3-ITDNEG vs. NPM1WT/FLT3-ITDNEG patients in the age groups <55 years (not estimable vs. 155 mo. p=0.066), 65-75 years (13 vs. 13.1 mo., p=0.46), and >75 years (3.3 vs. 5.2 mo., p=0.32). NPM1MT patients with lesser co-occurring mutations had a greater OS [≤3 vs. >3 total mutations, 51 vs. 18 mo., p=0.046]. The median age was 60.5 years compared to 66.4 years in the two populations, respectively (p=0.001). The most common associated mutations in <65 vs. >65 year with NPM1MT were DNMT3A (38% vs. 43%, p=0.27), IDH2 (23% vs. 25%, p=0.61), TET2 (13% vs. 27%, p=0.0001), FLT3-ITD (21% vs. 16%, p=0.16), IDH1 (19% vs. 15%, p=0.25), NRAS (13% vs. 7%, p=0.03), SRSF2 (5% vs. 15%, p=0.0002), WT1 (10% vs. 4%, p=0.01), PTPN11 (5% vs. 8%, p=0.18), CEBPA (5% vs. 6%, p=0.63), and ASXL1 (1% vs 7%, p=0.0006). In comparing dominant vs. non-dominant NPM1MT, OS was not different in NPM1MT/FLT3-ITDNEG patients [46.5 vs. 21 mo. P=0.42); however, there was a trend towards better survival in the NPM1MT/FLT3-ITDPOS group [OS not estimable vs. 22 mo., p=0.06 resp.]. In sum, extensive analysis of the age-stratified impact of NPM1MT in AML revealed intricate details of prognostic groups. We validated the prior findings of the not favorable impact of NPM1MT in older (>65 ys.) AML patients. We also found no difference in the frequently associated negatively impacting (such as FLT3) mutations. Since this dataset broadly represents patients treated outside clinical trials, it underscores whether they were undertreated or truly ineligible for intensive induction therapy. If the former is true, older NPM1MT AML should be stringently selected for aggressive therapeutic or targeted interventions, which could enhance their survival in this otherwise positive prognostic group. Our ongoing analysis, to be presented at the ASH meeting, will determine whether the less intense therapy may be the culprit and identify any subgroup that would respond better. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Menin Inhibitor Ziftomenib (KO-539) Synergizes with Agents Targeting Chromatin Regulation or Apoptosis and Sensitizes AML with MLL Rearrangement or NPM1 Mutation to Venetoclax

MLL1-rearranged (MLL-r) leukemias are dependent on the interaction of the adaptor protein menin with the oncogenic MLL (KMT2A) fusion protein. We previously demonstrated that NPM1 mutant (NPM1mut) acute myeloid leukemias (AMLs) exhibit a similar dependency on the menin interaction with wildtype MLL, a histone 3 lysine 4 methyltransferase. Pharmacologic inhibition of the menin-MLL interaction reverses MEIS1/HOX-driven leukemic gene expression, induces differentiation, and has anti-leukemic activity in preclinical NPM1mut and MLL-r AML models. Ziftomenib is currently assessed in clinical phase I/II trials and explorative efficacy against relapsed / refractory AML has been demonstrated. Here, we characterized the effects of ziftomenib on MLL-r and NPM1mut AML in detail and systematically screened for synergistic drug combination partners. First, we assessed the single-agent effects of ziftomenib on MLL-r (MOLM13, MV411, OCI-AML2) and NPM1mut (OCI-AML3) AML cell lines. Cellular assays demonstrated profound dose-dependent killing after seven days of treatment with IC50 values<25nM. HL60 and NB4 cells that are both wildtype for NPM1 and MLL served as negative controls and were not affected. Similar to previously characterized menin inhibitors (menin-i), ziftomenib uniformly induced transcriptional downregulation of MEIS1, PBX3, FLT3, and BCL2 in these leukemias, while genes upregulated upon ziftomenib were enriched for myelomonocytic differentiation processes. Ziftomenib-induced differentiation was also evidenced by cytology and upregulation of cell surface CD14 and CD11b. To identify synergistic combinations, we designed a screen assessing single and combined effects of ziftomenib and 37 targeted compounds (selected by confirmed preclinical or clinical activity against AML), each at four different concentrations and in a constant 1:4 ratio. We observed significant synergistic effects (combination index<0.5) of ziftomenib in MLL-r and NPM1mut AML, particularly pronounced for agents targeting chromatin regulation & DNA damage (e.g., LSD1, PRMT5, and PARP) as well as apoptosis & cell cycle (e.g. BCL2, MCL1, and CDK4/6). As we have demonstrated with other menin-i, ziftomenib induced transcriptional downregulation of FLT3 in the FLT3-mutant AML setting and also exhibited synergistic activity with the FLT3 inhibitor gilteritinib. As ziftomenib also uniformly downregulated BCL2, we assessed its combination with venetoclax in more detailed response assays and found synergistic growth inhibition compared to single treatment or vehicle control in all tested MLL-r and NPM1mut AMLs. While OCI-AML3 cells are not sensitive to venetoclax, the combination with ziftomenib was synergistic when using prolonged combined agent exposure (5 days) but not with standard 24-48h venetoclax treatment. shRNA-based knockdown of MEN1 (menin) also sensitized MOLM13 to venetoclax. Next, we found by Annexin V staining that the combination enhanced apoptosis compared to single agent treatment. Also, BCL2 homology (BH) 3 profiling revealed that ziftomenib treated MLL-r and NPM1mut AML cells exhibited a significantly enhanced cytochrome c release in response to BIM, PUMA, BAD, and MS1 peptides. These findings suggest that ziftomenib-induced apoptotic priming may contribute to synergistic effects with venetoclax in these leukemias. Next, we assessed the combination in a human MV411-derived leukemic xenograft model in vivo. We found significantly reduced leukemia burden defined by bone marrow engraftment after 14 days of treatment compared to the single agent or vehicle control animals. Single agent and combination assessment of four primary human NPM1mut AML patient samples in a human stroma cell co-culture model also showed the most pronounced anti-leukemic effects for the ziftomenib-venetoclax combination compared to all other controls. In summary, we demonstrated that ziftomenib has significant activity against MLL-r and NPM1mut AML and synergizes with certain agents targeting chromatin regulation and apoptosis. The combination of ziftomenib with venetoclax is particularly synergistic and already available for clinical trial assessment.