Role for PKC δ in Fenretinide-Mediated Apoptosis in Lymphoid Leukemia Cells

The synthetic Vitamin A analog fenretinide is a promising chemotherapeutic agent. In the current paper, the role of PKC δ was examined in fenretinide-induced apoptosis in lymphoid leukemia cells. Levels of proapoptotic cleaved PKC δ positively correlated with drug sensitivity. Fenretinide promoted reactive oxygen species (ROS) generation. The antioxidant Vitamin C prevented fenretinide-induced PKC δ cleavage and protected cells from fenretinide. Suppression of PKC δ expression by shRNA sensitized cells to fenretinide-induced apoptosis possibly by a mechanism involving ROS production. A previous study demonstrated that fenretinide promotes degradation of antiapoptotic MCL-1 in ALL cells via JNK. Now we have found that fenretinide-induced MCL-1 degradation may involve PKC δ as cleavage of the kinase correlated with loss of MCL-1 even in cells when JNK was not activated. These results suggest that PKC δ may play a complex role in fenretinide-induced apoptosis and may be targeted in antileukemia strategies that utilize fenretinide

Anexelekto/MER tyrosine kinase inhibitor ONO-7475 arrests growth and kills FMS-like tyrosine kinase 3-internal tandem duplication mutant acute myeloid leukemia cells by diverse mechanisms

Nearly one-third of patients with acute myeloid leukemia have FMS-like tyrosine kinase 3 mutations and thus have poor survival prospects. Receptor tyrosine kinase anexelekto is critical for FMS-like tyrosine kinase 3 signaling and participates in FMS-like tyrosine kinase 3 inhibitor resistance mechanisms. Thus, strategies targeting anexelekto could prove useful for acute myeloid leukemia therapy. ONO-7475 is an inhibitor with high specificity for anexelekto and MER tyrosine kinase. Herein, we report that ONO-7475 potently arrested growth and induced apoptosis in acute myeloid leukemia with internal tandem duplication mutation of FMS-like tyrosine kinase 3. MER tyrosine kinase-lacking MOLM13 cells were sensitive to ONO-7475, while MER tyrosine kinase expressing OCI-AML3 cells were resistant, suggesting that the drug acts via anexelekto in acute myeloid leukemia cells. Reverse phase protein analysis of ONO-7475 treated cells revealed that cell cycle regulators like cyclin dependent kinase 1, cyclin B1, polo-like kinase 1, and retinoblastoma were suppressed. ONO-7475 suppressed cyclin dependent kinase 1, cyclin B1, polo-like kinase 1 gene expression suggesting that anexelekto may regulate the cell cycle, at least in part, via transcriptional mechanisms. Importantly, ONO-7475 was effective in a human FMS-like tyrosine kinase 3 with internal tandem duplication mutant murine xenograft model. Mice fed a diet containing ONO-7475 exhibited significantly longer survival and, interestingly, blocked leukemia cell infiltration in the liver. In summary, ONO-7475 effectively kills acute myeloid leukemia cells in vitro and in vivo by mechanisms that involve disruption of diverse survival and proliferation pathways

Combined inhibition of BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance of TP53-mutant acute myeloid leukemia to individual BH3 mimetics

TP53-mutant acute myeloid leukemia (AML) respond poorly to currently available treatments, including venetoclax-based drug combinations and pose a major therapeutic challenge. Analyses of RNA sequencing and reverse phase protein array datasets revealed significantly lower BAX RNA and protein levels in TP53-mutant compared to TP53-wild-type (WT) AML, a finding confirmed in isogenic CRISPR-generated TP53-knockout and -mutant AML. The response to either BCL-2 (venetoclax) or MCL-1 (AMG176) inhibition was BAX-dependent and much reduced in TP53-mutant compared to TP53-WT cells, while the combination of two BH3 mimetics effectively activated BAX, circumventing survival mechanisms in cells treated with either BH3 mimetic, and synergistically induced cell death in TP53-mutant AML and stem/progenitor cells. The BH3 mimetic-driven stress response and cell death patterns after dual inhibition were largely independent of TP53 status and affected by apoptosis induction. Co-targeting, but not individual targeting of BCL-2 and MCL-1 in mice xenografted with TP53-WT and TP53-R248W Molm13 cells suppressed both TP53-WT and TP53-mutant cell growth and significantly prolonged survival. Our results demonstrate that co-targeting BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance to individual BH3 mimetics in TP53-mutant cells, thus shifting cell fate from survival to death in TP53-deficient and -mutant AML. This concept warrants clinical evaluation

C-MYC Targeting by Degradation: Novel Dual c-Myc/GSPT1 Degrader GT19715 Exerts Profound Cell Kill in vitro and in vivo in Acute Myeloid Leukemia and Lymphomas

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Targeting MCL-1 dysregulates cell metabolism and leukemia-stroma interactions and re-sensitizes acute myeloid leukemia to BCL-2 inhibition

MCL-1 and BCL-2 are both frequently overexpressed in acute myeloid leukemia (AML) and critical for the survival of AML cells and AML stem cells. MCL-1 is a key factor in venetoclax resistance. Using genetic and pharmacological approaches, we discovered that MCL-1 regulates leukemia cell bioenergetics and carbohydrate metabolisms, including the TCA cycle, glycolysis and pentose phosphate pathway and modulates cell adhesion proteins and leukemia-stromal interactions. Inhibition of MCL-1 sensitizes to BCL-2 inhibition in AML cells and AML stem/progenitor cells, including those with intrinsic and acquired resistance to venetoclax through cooperative release of pro-apoptotic BIM, BAX, and BAK from binding to anti-apoptotic BCL- 2 proteins and inhibition of cell metabolism and key stromal microenvironmental mechanisms. The combined inhibition of MCL-1 by MCL-1 inhibitor AZD5991 or CDK9 inhibitor AZD4573 and BCL-2 by venetoclax greatly extended survival of mice bearing patient-derived xenografts established from an AML patient who acquired resistance to venetoclax/decitabine. These results demonstrate that co-targeting MCL-1 and BCL-2 improves the efficacy of and overcomes pre-existing and acquired resistance to BCL-2 inhibition. Activation of metabolomic pathways and leukemia-stroma interactions are newly discovered functions of MCL-1 in AML, which are independent from canonical regulation of apoptosis by MCL-1. Our data provide new mechanisms of synergy and a rationale for co-targeting MCL-1 and BCL-2 clinically in patients with AML and potentially other cancers

Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia

SummaryBCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered

Distinct protein signatures of acute myeloid leukemia bone marrow-derived stromal cells are prognostic for patient survival

Mesenchymal stromal cells (MSC) support acute myeloid leukemia (AML) cell survival in the bone marrow (BM) microenvironment. Protein expression profiles of AML-derived MSC are unknown. Reverse phase protein array analysis was performed to compare expression of 151 proteins from AML-MSC (n=106) with MSC from healthy donors (n=71). Protein expression differed significantly between the two groups with 19 proteins over-expressed in leukemia stromal cells and 9 over-expressed in normal stromal cells. Unbiased hierarchical clustering analysis of the samples using these 28 proteins revealed three protein constellations whose variation in expression defined four MSC protein expression signatures: Class 1, Class 2, Class 3, and Class 4. These cell populations appear to have clinical relevance. Specifically, patients with Class 3 cells have longer survival and remission duration compared to other groups. Comparison of leukemia MSC at first diagnosis with those obtained at salvage (i.e. relapse/refractory) showed differential expression of 9 proteins reflecting a shift toward osteogenic differentiation. Leukemia MSC are more senescent compared to their normal counterparts, possibly due to the overexpressed p53/p21 axis as confirmed by high β-galactosidase staining. In addition, overexpression of BCL-XL in leukemia MSC might give survival advantage under conditions of senescence or stress and overexpressed galectin-3 exerts profound immunosuppression. Together, our findings suggest that the identification of specific populations of MSC in AML patients may be an important determinant of therapeutic response

Enhanced TP53 Reactivation Disrupts MYC Transcriptional Program and Overcomes Venetoclax Resistance in Acute Myeloid Leukemias

View full abstracthttps://openworks.mdanderson.org/leading-edge/1019/thumbnail.jp

Enhanced TP53 reactivation disrupts MYC transcriptional program and overcomes venetoclax resistance in acute myeloid leukemias

The tumor suppressor TP53 is frequently inactivated in a mutation-independent manner in cancers and is reactivated by inhibiting its negative regulators. We here cotarget MDM2 and the nuclear exporter XPO1 to maximize transcriptional activity of p53. MDM2/XPO1 inhibition accumulated nuclear p53 and elicited a 25- to 60-fold increase of its transcriptional targets. TP53 regulates MYC, and MDM2/XPO1 inhibition disrupted the c-MYC-regulated transcriptome, resulting in the synergistic induction of apoptosis in acute myeloid leukemia (AML). Unexpectedly, venetoclax-resistant AMLs express high levels of c-MYC and are vulnerable to MDM2/XPO1 inhibition in vivo. However, AML cells persisting after MDM2/XPO1 inhibition exhibit a quiescence- and stress response-associated phenotype. Venetoclax overcomes that resistance, as shown by single-cell mass cytometry. The triple inhibition of MDM2, XPO1, and BCL2 was highly effective against venetoclax-resistant AML in vivo. Our results propose a novel, highly translatable therapeutic approach leveraging p53 reactivation to overcome nongenetic, stress-adapted venetoclax resistance

Inhibition of translation initiation factor eIF4a inactivates heat shock factor 1 (HSF1) and exerts anti-leukemia activity in AML

Eukaryotic initiation factor 4A (eIF4A), the enzymatic core of the eIF4F complex essential for translation initiation, plays a key role in the oncogenic reprogramming of protein synthesis, and thus is a putative therapeutic target in cancer. As important component of its anticancer activity, inhibition of translation initiation can alleviate oncogenic activation of HSF1, a stress-inducible transcription factor that enables cancer cell growth and survival. Here, we show that primary acute myeloid leukemia (AML) cells exhibit the highest transcript levels of eIF4A1 compared to other cancer types. eIF4A inhibition by the potent and specific compound rohinitib (RHT) inactivated HSF1 in these cells, and exerted pronounced in vitro and in vivo anti-leukemia effects against progenitor and leukemia-initiating cells, especially those with FLT3-internal tandem duplication (ITD). In addition to its own anti-leukemic activity, genetic knockdown of HSF1 also sensitized FLT3-mutant AML cells to clinical FLT3 inhibitors, and this synergy was conserved in FLT3 double-mutant cells carrying both ITD and tyrosine kinase domain mutations. Consistently, the combination of RHT and FLT3 inhibitors was highly synergistic in primary FLT3-mutated AML cells. Our results provide a novel therapeutic rationale for co-targeting eIF4A and FLT3 to address the clinical challenge of treating FLT3-mutant AML.R01 CA175744 - NCI NIH HHS; R35 GM118173 - NIGMS NIH HHS; P30 CA016672 - NCI NIH HHSPublished versionSupporting documentationAccepted manuscrip