Role of the Crosstalk B:Neoplastic T Follicular Helper Cells in the Pathobiology of Nodal T Follicular Helper Cell Lymphomas.

Angioimmunoblastic T-cell lymphoma (AITL), the most common form of peripheral T-cell lymphoma, originates from follicular helper T (Tfh) cells and is notably resistant to current treatments. The disease progression and maintenance, at least in early stages, are driven by a complex interplay between neoplastic Tfh and clusters of B-cells within the tumor microenvironment, mirroring the functional crosstalk observed inside germinal centers. This interaction is further complicated by recurrent mutations, such as TET2 and DNMT3A, which are present in both Tfh cells and B-cells. These findings suggest that the symbiotic relationship between these 2 cell types could represent a therapeutic vulnerability. This review examines the key components and signaling mechanisms involved in the synapses between B-cells and Tfh cells, emphasizing their significant role in the pathobiology of AITL and potential as therapeutic targets.

LP-118 is a novel B-cell lymphoma 2 / extra-large inhibitor that demonstrates efficacy in models of venetoclax-resistant chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) respond well to initial treatment with the Bcell lymphoma 2 (BCL2) inhibitor venetoclax. Upon relapse, they often retain sensitivity to BCL2 targeting, but durability of response remains a concern. We hypothesize that targeting both BCL2 and B-cell lymphoma-extra large (BCLXL) will be a successful strategy to treat CLL, including for patients who relapse on venetoclax. To test this hypothesis, we conducted a pre-clinical investigation of LP-118, a highly potent inhibitor of BCL2 with moderate BCLXL inhibition to minimize platelet toxicity. This study demonstrated that LP-118 induces efficient BAK activation, cytochrome C release, and apoptosis in both venetoclax naïve and resistant CLL cells. Significantly, LP-118 is effective in cell lines expressing the BCL2 G101V mutation and in cells expressing BCLXL but lacking BCL2 dependence. Using an immunocompetent mouse model, Eµ-TCL1, LP-118 demonstrates low platelet toxicity, which hampered earlier BCLXL inhibitors. Finally, LP-118 in the RS4;11 and OSU-CLL xenograft models results in decreases in tumor burden and survival advantage, respectively. These results provide a mechanistic rationale for the evaluation of LP-118 for the treatment of venetoclax responsive and relapsed CLL.

Long-read single-cell RNA sequencing enables the study of cancer subclone-specific genotype and phenotype in chronic lymphocytic leukemia.

Bruton's tyrosine kinase (BTK) inhibitors are effective for the treatment of chronic lymphocytic leukemia (CLL) due to BTK's role in B cell survival and proliferation. Treatment resistance is most commonly caused by the emergence of the hallmark BTKC481S mutation that inhibits drug binding. In this study, we aimed to investigate whether the presence of additional CLL driver mutations in cancer subclones harboring a BTKC481S mutation accelerates subclone expansion. In addition, we sought to determine whether BTK-mutated subclones exhibit distinct transcriptomic behavior when compared to other cancer subclones. To achieve these goals, we employ our recently published method (Qiao et al. 2024) that combines bulk DNA sequencing and single-cell RNA sequencing (scRNA-seq) data to genotype individual cells for the presence or absence of subclone-defining mutations. While the most common approach for scRNA-seq includes short-read sequencing, transcript coverage is limited due to the vast majority of the reads being concentrated at the priming end of the transcript. Here, we utilized MAS-seq, a long-read scRNAseq technology, to substantially increase transcript coverage across the entire length of the transcripts and expand the set of informative mutations to link cells to cancer subclones in six CLL patients who acquired BTKC481S mutations during BTK inhibitor treatment. We found that BTK-mutated subclones often acquire additional mutations in CLL driver genes, leading to faster subclone proliferation. When examining subclone-specific gene expression, we found that in one patient, BTK-mutated subclones are transcriptionally distinct from the rest of the malignant B cell population with an overexpression of CLL-relevant genes.

VIP152 is a selective CDK9 inhibitor with pre-clinical in vitro and in vivo efficacy in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is effectively treated with targeted therapies including Bruton tyrosine kinase inhibitors and BCL2 antagonists. When these become ineffective, treatment options are limited. Positive transcription elongation factor complex (P-TEFb), a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, functions to regulate short half-life transcripts by phosphorylation of RNA Polymerase II (POLII). These transcripts are frequently dysregulated in hematologic malignancies; however, therapies targeting inhibition of P-TEFb have not yet achieved approval for cancer treatment. VIP152 kinome profiling revealed CDK9 as the main enzyme inhibited at 100 nM, with over a 10-fold increase in potency compared with other inhibitors currently in development for this target. VIP152 induced cell death in CLL cell lines and primary patient samples. Transcriptome analysis revealed inhibition of RNA degradation through the AU-Rich Element (ARE) dysregulation. Mechanistically, VIP152 inhibits the assembly of P-TEFb onto the transcription machinery and disturbs binding partners. Finally, immune competent mice engrafted with CLL-like cells of Eµ-MTCP1 over-expressing mice and treated with VIP152 demonstrated reduced disease burden and improvement in overall survival compared to vehicle-treated mice. These data suggest that VIP152 is a highly selective inhibitor of CDK9 that represents an attractive new therapy for CLL.

HDAC1 regulates the chromatin landscape to control transcriptional dependencies in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a quiescent B-cell malignancy that depends on transcriptional dysregulation for survival. The histone deacetylases are transcriptional regulators whose role within the regulatory chromatin and consequence on the CLL transcriptome is poorly characterized. Here, we profiled and integrated the genome-wide occupancy of HDAC1, BRD4, H3K27Ac, and H3K9Ac signals with chromatin accessibility, Pol2 occupancy, and target expression signatures in CLL cells. We identified that when HDAC1 was recruited within super-enhancers (SEs) marked by acetylated H3K27 and BRD4, it functioned as a transcriptional activator that drove the de novo expression of select genes to facilitate survival and progression in CLL. Targeting HDACs reduced BRD4 and Pol2 engagement to downregulate the transcript and proteins levels of specific oncogenic driver genes in CLL such as BLK, a key mediator of the B-cell receptor pathway, core transcription factors such as PAX5 and IKZF3, and the antiapoptotic gene, BCL2. Concurrently, HDAC1, when recruited in the absence of SEs, repressed target gene expression. HDAC inhibition reversed silencing of a defined set of protein-coding and noncoding RNA genes. We focused on a specific set of microRNA genes and showed that their upregulation was inversely correlated with the expression of CLL-specific survival, transcription factor, and signaling genes. Our findings identify that the transcriptional activator and repressor functions of HDACs cooperate within the same tumor to establish the transcriptional dependencies essential for survival in CLL.

Preclinical evaluation of combination nemtabrutinib and venetoclax in chronic lymphocytic leukemia.

Inhibitors of B cell receptor (BCR) signaling such as the Bruton's tyrosine kinase (BTK) inhibitors are effective therapeutics for chronic lymphocytic leukemia (CLL). The first-in-class covalent BTK inhibitor, ibrutinib, produces durable responses in most CLL patients; however, complete responses are only observed in a minority of patients. B cell lymphoma 2 (BCL2), an anti-apoptotic protein that contributes to CLL cell survival, has also been investigated as a therapeutic target. The BCL2 inhibitor venetoclax is effective in patients with CLL and can produce undetectable minimal residual disease, allowing discontinuation of therapy. In combination, ibrutinib and venetoclax have shown preclinical synergy and clinical efficacy. Nemtabrutinib is a next generation, reversible inhibitor of BTK that potently inhibits BCR signaling in treatment-naïve and ibrutinib-refractory CLL cells ex vivo. The clinical efficacy of combining BTK inhibitors with BCL2 inhibitors motivated us to evaluate the novel combination of nemtabrutinib and venetoclax. In vitro studies show that nemtabrutinib and venetoclax are not antagonistic to each other. In an adoptive transfer CLL mouse model, mice treated with nemtabrutinib and venetoclax had prolonged survival compared to mice treated with ibrutinib and venetoclax. Our preclinical studies further validate the combination of BTK inhibitors with venetoclax and justify further investigation of combining nemtabrutinib with venetoclax in CLL.

Targeting OXPHOS de novo purine synthesis as the nexus of FLT3 inhibitor-mediated synergistic antileukemic actions.

Using a genome-wide CRISPR screen, we identified CDK9, DHODH, and PRMT5 as synthetic lethal partners with gilteritinib treatment in fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) acute myeloid leukemia (AML) and genetically and pharmacologically validated their roles in gilteritinib sensitivity. The presence of FLT3-ITD is associated with an increase in anaerobic glycolysis, rendering leukemia cells highly sensitive to inhibition of glycolysis. Supportive of this, our data show the enrichment of single guide RNAs targeting 28 glycolysis-related genes upon gilteritinib treatment, suggesting that switching from glycolysis to oxidative phosphorylation (OXPHOS) may represent a metabolic adaption of AML in gilteritinib resistance. CDK9i/FLT3i, DHODHi/FLT3i, and PRMT5i/FLT3i pairs mechanistically converge on OXPHOS and purine biosynthesis blockade, implying that targeting the metabolic functions of these three genes and/or proteins may represent attractive strategies to sensitize AML to gilteritinib treatment. Our findings provide the basis for maximizing therapeutic impact of FLT3-ITD inhibitors and a rationale for a clinical trial of these novel combinations.

TP53-altered chronic lymphocytic leukemia treated with firstline Bruton's tyrosine kinase inhibitor-based therapy: A retrospective analysis.

Long-term follow up of prospective studies has shown that continuous Bruton's tyrosine kinase inhibitor (BTKi) therapy leads to durable remissions in previously untreated patients with TP53-altered chronic lymphocytic leukemia (CLL); however, it is unknown how variant allele frequency (VAF) of TP53 mutation (TP53-m) or percentage of cells with deletion of chromosome 17p [del(17p)] influences efficacy of firstline BTKi. We performed a retrospective analysis of 130 patients with CLL with baseline del(17p) and/or TP53-m treated with BTKi with or without the BCL2 inhibitor venetoclax (VEN) and with or without CD20 antibody in the firstline setting. A total of 104/130 (80%) patients had del(17p). TP53-m was noted in 89/110 (81%) patients tested; there were 101 unique TP53-m with an available VAF. The 4-year progression-free survival (PFS) and overall survival (OS) rates were 72.9% and 83.6%. No baseline characteristics including IGHV mutation status and number of TP53 alterations were associated with significant differences in PFS or OS, though a trend toward shorter PFS with increasing karyotypic complexity (hazard ratio 1.08, p = .066) was observed. Del(17p) was identified in <25% of cells in 26/104 (25%) of patients, and 28/101 (28%) of TP53-m were low-burden with a VAF of <10%; outcomes of these patients were similar to those with high-burden lesions. This study suggests that low-burden TP53 alterations should not be ignored when assessing genomic risk in CLL in the era of targeted therapy.

Evaluation of allogeneic and autologous membrane-bound IL-21-expanded NK cells for chronic lymphocytic leukemia therapy.

Successes with anti-CD20 antibodies in chronic lymphocytic leukemia (CLL) and enhanced activity of Fc-engineered vs unmodified antibody therapy suggest a potentially impactful role for natural killer (NK) cells and other innate immune cells in controlling this disease. Stimulated NK cells have shown promise as a cellular therapy, but their application has been constrained by limited expansion capacity and low cytotoxic activity against CLL cells. Here, we demonstrate that both healthy donor-derived and CLL patient-derived NK cells expand rapidly when stimulated with feeder cells expressing membrane-bound interleukin-21 (mbIL-21) and have potent cytotoxic activity against allogeneic or autologous CLL cells. Combination with anti-CD20 antibodies significantly enhances NK recognition and killing of CLL targets. As any CLL immune therapy would likely be given in combination, we assess commonly used treatments and demonstrate that ibrutinib has mixed suppressive and protective effects on expanded NK cells, whereas expanded NKs are highly resistant to venetoclax. We demonstrate efficacy in vivo in 2 xenograft mouse models of human CLL that support building upon a regimen of venetoclax and obinutuzumab with mbIL-21-expanded NK cells. Collectively, these data support development of mbIL-21-expanded NKs combined with the CD20 antibody obinutuzumab and venetoclax in the treatment of CLL.

Disruption of DNA Repair and Survival Pathways through Heat Shock Protein Inhibition by Onalespib to Sensitize Malignant Gliomas to Chemoradiation Therapy.

PURPOSE: Proficient DNA repair by homologous recombination (HR) facilitates resistance to chemoradiation in glioma stem cells (GSC). We evaluated whether compromising HR by targeting HSP90, a molecular chaperone required for the function of key HR proteins, using onalespib, a long-acting, brain-penetrant HSP90 inhibitor, would sensitize high-grade gliomas to chemoradiation in vitro and in vivo. EXPERIMENTAL DESIGN: The ability of onalespib to deplete HR client proteins, impair HR repair capacity, and sensitize glioblastoma (GBM) to chemoradiation was evaluated in vitro in GSCs, and in vivo using zebrafish and mouse intracranial glioma xenograft models. The effects of HSP90 inhibition on the transcriptome and cytoplasmic proteins was assessed in GSCs and in ex vivo organotypic human glioma slice cultures. RESULTS: Treatment with onalespib depleted CHK1 and RAD51, two key proteins of the HR pathway, and attenuated HR repair, sensitizing GSCs to the combination of radiation and temozolomide (TMZ). HSP90 inhibition reprogrammed the transcriptome of GSCs and broadly altered expression of cytoplasmic proteins including known and novel client proteins relevant to GSCs. The combination of onalespib with radiation and TMZ extended survival in a zebrafish and a mouse xenograft model of GBM compared with the standard of care (radiation and TMZ) or onalespib with radiation. CONCLUSIONS: The results of this study demonstrate that targeting HR by HSP90 inhibition sensitizes GSCs to radiation and chemotherapy and extends survival in zebrafish and mouse intracranial models of GBM. These results provide a preclinical rationale for assessment of HSP90 inhibitors in combination with chemoradiation in patients with GBM.

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the nicotinamide adenine dinucleotide (NAD) salvage pathway, to target glioma heterogeneity through mitochondrial oxidative stress.

BACKGROUND: Tumor-specific metabolic processes essential for cell survival are promising targets to potentially circumvent intratumoral heterogeneity, a major resistance factor in gliomas. Tumor cells preferentially using nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage pathway for synthesis of NAD, a critical cofactor for diverse biological processes including cellular redox reactions, energy metabolism, and biosynthesis. NAMPT is overexpressed in most malignancies, including gliomas, and can serve as a tumor-specific target. METHODS: Effects of pharmacological inhibition of NAMPT on cellular oxygen consumption rate, extracellular acidification, mitochondrial respiration, cell proliferation, invasion, and survival were assessed through in vitro and ex vivo studies on genetically heterogeneous glioma cell lines, glioma stem-like cells (GSCs), and mouse and human ex vivo organotypic glioma slice culture models. RESULTS: Pharmacological inhibition of the NAD salvage biosynthesis pathway using a highly specific inhibitor, KPT-9274, resulted in the reduction of NAD levels and related downstream metabolites, inhibited proliferation, and induced apoptosis in vitro in cell lines and ex vivo in human glioma tissue. These effects were mediated by mitochondrial dysfunction, DNA damage, and increased oxidative stress leading to apoptosis in GSCs independent of genotype, IDH status, or MGMT promoter methylation status. Conversely, NAMPT inhibition had minimal in vitro effects on normal human astrocytes (NHA) and no apparent in vivo toxicity in non-tumor-bearing mice. CONCLUSIONS: Pharmacological NAMPT inhibition by KPT9274 potently targeted genetically heterogeneous gliomas by activating mitochondrial dysfunction. Our preclinical results provide a rationale for targeting the NAMPT-dependent alternative NAD biosynthesis pathway as a novel clinical strategy against gliomas.

Anti-tumor NAMPT inhibitor, KPT-9274, mediates gender-dependent murine anemia and nephrotoxicity by regulating SIRT3-mediated SOD deacetylation.

KPT-9274 is a phase 1 first-in-class dual PAK4/NAMPT inhibitor for solid tumor and non-Hodgkin's lymphoma. It demonstrates pre-clinical efficacy toward a broad spectrum of acute myeloid leukemia (AML) subtypes by inhibiting NAMPT-dependent NAD+ production. NAMPT is the rate-limiting enzyme in the salvage metabolic pathway leading to NAD+ generation. Tumor cells which are deficient in de novo pathway enzyme NAPRT1 are addicted to NAMPT. In clinical trials, treatment with NAMPT inhibitors resulted in dose-limiting toxicities. In order to dissect the mechanism of toxicity, mice were treated with KPT-9274 and resulting toxicities were characterized histopathologically and biochemically. KPT-9274 treatment caused gender-dependent stomach and kidney injuries and anemia. Female mice treated with KPT-9274 had EPO deficiency and associated impaired erythropoiesis. KPT-9274 treatment suppressed SIRT3 expression and concomitantly upregulated acetyl-manganese superoxide dismutase (MnSOD) in IMCD3 cells, providing a mechanistic basis for observed kidney toxicity. Importantly, niacin supplementation mitigated KPT-9274-caused kidney injury and EPO deficiency without affecting its efficacy. Altogether, our study delineated the mechanism of KPT-9274-mediated toxicity and sheds light onto developing strategies to improve the tolerability of this important anti-AML inhibitor.

Targeting DNA Damage Repair Functions of Two Histone Deacetylases, HDAC8 and SIRT6, Sensitizes Acute Myeloid Leukemia to NAMPT Inhibition.

PURPOSE: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPTi) are currently in development, but may be limited as single-agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPTis required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPTi, KPT-9274. EXPERIMENTAL DESIGN: Cell lines and primary cells were analyzed for cell viability, self-renewal, and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. In vivo efficacy of combination therapy was evaluated with a xenograft model. RESULTS: We identified two histone deacetylases (HDAC), HDAC8 and SIRT6, whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 cotreatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising in vivo efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 cotreatment resulted in synergistic attenuation of homologous recombination and nonhomologous end joining pathways in cell lines and leukemia-initiating cells. CONCLUSIONS: Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity toward normal cells, providing a rationale for a novel-novel combination-based treatment for AML.

Preclinical evaluation of the Hsp90 inhibitor SNX-5422 in ibrutinib resistant CLL.

B-cell receptor (BCR) antagonists such as the BTK inhibitor ibrutinib have proven to effectively target chronic lymphocytic leukemia (CLL) tumor cells, leading to impressive response rates in these patients. However patients do still relapse on ibrutinib, and the progressive disease is often quite aggressive requiring immediate treatment. Several strategies are being pursued to treat patients who relapse on ibrutinib therapy. As the most common form of relapse is the development of a mutant form of BTK which limits ibrutinib binding, agents which lead to degradation of the BTK protein are a promising strategy. Our study explores the efficacy of the Hsp90 inhibitor, SNX-5422, in CLL. The SNX Hsp90 inhibitor was effective in primary CLL cells, as well as B-cell lines expressing either BTK wild type or C481 mutant BTK, which has been identified as the primary resistance mechanism to ibrutinib in CLL patients. Furthermore the combination of SNX-5422 and ibrutinib provided a remarkable in vivo survival benefit in the Eµ-TCL1 mouse model of CLL compared to the vehicle or single agent groups (51 day median survival in the vehicle and ibrutinib groups versus 100 day median survival in the combination). We report here preclinical data suggesting that the Hsp90 inhibitor SNX-5422, which has been pursued in clinical trials in both solid tumor and hematological malignancies, is a potential therapy for ibrutinib resistant CLL.

Explaining Gene Expression Using Twenty-One MicroRNAs.

The transcriptome of a tumor contains detailed information about the disease. Although advances in sequencing technologies have generated larger data sets, there are still many questions about exactly how the transcriptome is regulated. One class of regulatory elements consists of microRNAs (or miRs), many of which are known to be associated with cancer. To better understand the relationships between miRs and cancers, we analyzed ∼9000 samples from 32 cancer types studied in The Cancer Genome Atlas. Our feature reduction algorithm found evidence for 21 biologically interpretable clusters of miRs, many of which were statistically associated with a specific type of cancer. Moreover, the clusters contain sufficient information to distinguish between most types of cancer. We then used linear models to measure, genome-wide, how much variation in gene expression could be explained by the 21 average expression values ("scores") of the clusters. Based on the ∼20,000 per-gene R2 values, we found that (1) mean differences between tissues of origin explain about 36% of variation; (2) the 21 miR cluster scores explain about 30% of the variation; and (3) combining tissue type with the miR scores explained about 56% of the total genome-wide variation in gene expression. Our analysis of poorly explained genes shows that they are enriched for olfactory receptor processes, sensory perception, and nervous system processing, which are necessary to receive and interpret signals from outside the organism. Therefore, it is reasonable for those genes to be always active and not get downregulated by miRs. In contrast, highly explained genes are characterized by genes translating to proteins necessary for transport, plasma membrane, or metabolic processes that are heavily regulated processes inside the cell. Other genetic regulatory elements such as transcription factors and methylation might help explain some of the remaining variation in gene expression.

Eµ-TCL1xMyc: A Novel Mouse Model for Concurrent CLL and B-Cell Lymphoma.

PURPOSE: Aberrant Myc expression is a major factor in the pathogenesis of aggressive lymphoma, and these lymphomas, while clinically heterogeneous, often are resistant to currently available treatments and have poor survival. Myc expression can also be seen in aggressive lymphomas that are observed in the context of CLL, and we sought to develop a mouse model that could be used to study therapeutic strategies for aggressive lymphoma in the context of CLL. EXPERIMENTAL DESIGN: We crossed the Eµ-TCL1 mouse model with the Eµ-Myc mouse model to investigate the clinical phenotype associated with B-cell-restricted expression of these oncogenes. The resulting malignancy was then extensively characterized, from both a clinical and biologic perspective. RESULTS: Eµ-TCL1xMyc mice uniformly developed highly aggressive lymphoid disease with histologically, immunophenotypically, and molecularly distinct concurrent CLL and B-cell lymphoma, leading to a significantly reduced lifespan. Injection of cells from diseased Eµ-TCL1xMyc into WT mice established a disease similar to that in the double-transgenic mice. Both Eµ-TCL1xMyc mice and mice with disease after adoptive transfer failed to respond to ibrutinib. Effective and durable disease control was, however, observed by selective inhibition of nuclear export protein exportin-1 (XPO1) using a compound currently in clinical development for relapsed/refractory malignancies, including CLL and lymphoma. CONCLUSIONS: The Eµ-TCL1xMyc mouse is a new preclinical tool for testing experimental drugs for aggressive B-cell lymphoma, including in the context of CLL.

Selective targeting of NAMPT by KPT-9274 in acute myeloid leukemia.

Treatment options for acute myeloid leukemia (AML) remain extremely limited and associated with significant toxicity. Nicotinamide phosphoribosyltransferase (NAMPT) is involved in the generation of NAD+ and a potential therapeutic target in AML. We evaluated the effect of KPT-9274, a p21-activated kinase 4/NAMPT inhibitor that possesses a unique NAMPT-binding profile based on in silico modeling compared with earlier compounds pursued against this target. KPT-9274 elicited loss of mitochondrial respiration and glycolysis and induced apoptosis in AML subtypes independent of mutations and genomic abnormalities. These actions occurred mainly through the depletion of NAD+, whereas genetic knockdown of p21-activated kinase 4 did not induce cytotoxicity in AML cell lines or influence the cytotoxic effect of KPT-9274. KPT-9274 exposure reduced colony formation, increased blast differentiation, and diminished the frequency of leukemia-initiating cells from primary AML samples; KPT-9274 was minimally cytotoxic toward normal hematopoietic or immune cells. In addition, KPT-9274 improved overall survival in vivo in 2 different mouse models of AML and reduced tumor development in a patient-derived xenograft model of AML. Overall, KPT-9274 exhibited broad preclinical activity across a variety of AML subtypes and warrants further investigation as a potential therapeutic agent for AML.