Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in cancer. Herein, we utilize BRD4 profiling to identify critical pathways involved in pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is overexpressed in CLL and is enriched proximal to genes upregulated or expressed in CLL with known functions in disease pathogenesis and progression. These genes, including key members of the B-cell receptor (BCR) signaling pathway, provide a rationale for this therapeutic approach to identify new targets in alternative types of cancer. Additionally, we describe PLX51107, a structurally distinct BET inhibitor with novel and pharmacologic properties that emulates or exceeds the efficacy of BCR signaling agents in preclinical models of CLL. Herein, the discovery of the involvement of BRD4 in the core CLL transcriptional program provides a compelling rationale for clinical investigation of PLX51107 as epigenetic therapy in CLL and application of BRD4 profiling in other cancers. To date, functional studies of BRD4 in CLL are lacking. Through integrated genomic, functional, and pharmacologic analyses, we uncover the existence of BRD4-regulated core CLL transcriptional programs and present preclinical proof-of-concept studies validating BET inhibition as an epigenetic approach to target BCR signaling in CLL. .
Acute promyelocytic leukemia (APL) is characterized by the t(15;17) chromosomal translocation, which results in fusion of the retinoic acid receptor α (RARA) gene to another gene, most commonly promyelocytic leukemia (PML). The resulting fusion protein, PML-RARA, initiates APL, which is a subtype (M3) of acute myeloid leukemia (AML). In this report, we identify a gene expression signature that is specific to M3 samples; it was not found in other AML subtypes and did not simply represent the normal gene expression pattern of primary promyelocytes. To validate this signature for a large number of genes, we tested a recently developed high throughput digital technology (NanoString nCounter). Nearly all of the genes tested demonstrated highly significant concordance with our microarray data (P < 0.05). The validated gene signature reliably identified M3 samples in 2 other AML datasets, and the validated genes were substantially enriched in our mouse model of APL, but not in a cell line that inducibly expressed PML-RARA. These results demonstrate that nCounter is a highly reproducible, customizable system for mRNA quantification using limited amounts of clinical material, which provides a valuable tool for biomarker measurement in low-abundance patient samples.
Key Points• KPT-9274, via its protein target NAMPT, diminishes NAD 1 levels and cellular respiration, leading to cell death.• Orally bioavailable KPT-9274 exhibits targetspecific activity in cell lines and patientderived xenograft models of AML. 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 1 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 1 , 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.
Acute promyelocytic leukemia (APL) is initiated by the PML-RARA fusion oncogene and has a characteristic expression profile that includes high levels of the Notch ligand JAG1. In this study, we used a series of bioinformatic, in vitro, and in vivo assays to assess the role of Notch signaling in human APL samples, and in a PML-RARA knockin mouse model of APL (Ctsg-PML-RARA). We identified a Notch expression signature in both human primary APL cells and in Kit+Lin−Sca1+ (KLS) cells from pre-leukemic Ctsg-PML-RARA mice. Both genetic and pharmacologic inhibition of Notch signaling abrogated the enhanced self-renewal seen in hematopoietic stem/progenitor cells (HSPCs) from pre-leukemic Ctsg-PML-RARA mice, but had no influence on cells from age-matched wildtype mice. In addition, 6 of 9 murine APL tumors tested displayed diminished growth in vitro when Notch signaling was inhibited pharmacologically. Finally, we found that genetic inhibition of Notch signaling with a dominant negative MAML protein reduced APL growth in vivo in a subset of tumors. These findings expand the role of Notch signaling in hematopoietic diseases, and further define the mechanistic events important for PML-RARA-mediated leukemogenesis.
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.
Background: Our group previously reported a 16% incidence of HC after hematopoietic cell transplantation (HCT) involving mostly matched unrelated donors (85%) and ATG-containing regimens (80%) (Silva, Haematologica 2010). PT-Cy is an effective GVHD prophylaxis in haplo-or matched unrelated transplants. There is a paucity of information about the risk of HC in this setting. Objective: To retrospectively assess the incidence and clinical factors associated with HC in alloHCT recipients receiving PTCy as GVHD prophylaxis. Methods: A retrospective analysis of patients (pts.) who underwent HCT with PT-Cy at our center between 2012-2018 was performed. We stratified the study cohort (N=478) into HC (N=193, 40%) and Non-HC (N=285, 60%) to compare their characteristics and outcome. Association between HC and clinical variables was assessed using logistic regression modeling. Results: Pt's age, HCT-CI, # prior therapies, disease type and status were not statistically different between the 2 groups. However, the HC group pts. were more likely to have received more intense conditioning (Busulfan/Fludarabine(BF) +/-Clofarabine, or Thio; F/Melphalan (FM) 140+/-Thio or TBI vs. FM 100+/-TBI) (p=0.018), had haplo-transplants (p=0.018), BM as graft (p=0.021), and tacrolimus+mycophenolate mofetil (MMF) (p=0.043). Delayed ANC engraftment (p=0.032) and time to achieve an absolute lymphocyte count >500 was noted in HC group (p=0.014). 54% of HC pts. had grade 2-4 cystitis and BK virus was isolated in 87% of cases. CMV viremia was higher in HC group compared to Non-HC group (p<0.001). HC pts. experienced a higher # of hospitalizations (p=0.012) and longer hospital stay (p=0.06) days compared to Non-HC pts. Additionally, HC pts. were more likely to experience grade II-IV aGVHD compared to Non-HC pts (p=0.005). In multivariable analysis, conditioning intensity remained significant [HR 2.27 (95% CI: 1.28, 4.01); p=0.005]. Conclusion: Our study showed a 40% incidence of HC after HCT recipients receiving PT-Cy. The intensity of the conditioning regimen is an independent predictive factor for developing HC. A formal comparison to ATG-containing regimens is in progress.
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