Steven Grant

Clinical, molecular, and cytogenetic characteristics of patient samples (CD45dim SS low population) exposed to ABT-199 ± INK-128

Clinical, molecular, and cytogenetic characteristics of patient samples (CD34+CD38-CD123+ population) exposed to ABT-199 ± INK-128

Summary: Histone deacetylase inhibitors (HDACIs) are approved for T‐cell lymphoma (TCL) treatment, but resistance represents a major problem. Recent encouraging clinical activity of the geranylgeranyl transferase inhibitor GGTI‐2418 (PTX‐100) in TCL prompted us to investigate HDACI interactions. HH, H9 and Hut78 TCL‐cell co‐treatment with the HDACIs belinostat or romidepsin with the GGTI‐2418 methyl ester pro‐drug GGTI‐2417 (GGTI) synergistically increased cell death. Synergism was mediated at least in part by AKT (Protein Kinase B) inhibition as demonstrated by (a) the HDACI/GGTI combination markedly decreased p‐AKT levels; (b) ectopic expression of constitutively active (myristoylated) AKT conferred significant resistance of cells to GGTI/HDACI; (c) highly HDACI‐resistant HH and H9 cells expressed high basal levels of phosphorylated AKT that were downregulated by GGTI/HDACI co‐administration, leading to cell death induction; (d) shRNA knockdown of AKT in HDACI‐resistant cells restored HDACI/GGTI sensitivity. Finally, flank xenograft models revealed that HDACI/GGTI combination treatment was significantly more effective than monotherapy in inhibiting in vivo TCL tumour growth. These findings argue that combined GGTI/HDACI exposure synergistically induces cell death and reduces HDACI resistance through an AKT‐dependent mechanism in TCL cells. In view of clinical results with GGTI‐2418, the HDACI/GGTI‐2418 combination strategy warrants consideration in T‐cell malignancies.

The importance of MCL-1 in leukemogenesis has prompted development of MCL-1 antagonists e.g., S63845, MIK665. However, their effectiveness in acute myeloid leukemia (AML) is limited by compensatory MCL-1 accumulation via the ubiquitin proteasome system. Here, we investigated mechanisms by which kinase inhibitors with Src inhibitory activity e.g., bosutinib (SKI-606) might circumvent this phenomenon. MCL-1 antagonist/SKI-606 co-administration synergistically induced apoptosis in diverse AML cell lines. Consistently, Src or MCL-1 knockdown with shRNA markedly sensitized cells to MCL-1 inhibitors or SKI-606 respectively, while ectopic MCL-1 expression significantly diminished apoptosis. Mechanistically, MCL-1 antagonist exposure induced MCL-1 up-regulation, an event blocked by Src inhibitors or Src shRNA knock-down. MCL-1 down-regulation was associated with diminished transcription and increased K48-linked degradative ubiquitination. Enhanced cell death depended functionally upon down-regulation of phosphorylated STAT3 (Tyr705/Ser727) and cytoprotective downstream targets c-Myc and BCL-xL, as well as BAX/BAK activation, and NOXA induction. Importantly, the Src/MCL-1 inhibitor regimen robustly killed primary AML cells, including primitive progenitors, but spared normal hematopoietic CD34⁺ cells and human cardiomyocytes. Notably, the regimen significantly improved survival in an MV4-11 cell xenograft model, while reducing tumor burden in two patient-derived xenograft (PDX) AML models and increased survival in a third. These findings argue that Src inhibitors such as SKI-606 potentiate MCL-1 antagonist anti-leukemic activity in vitro and in vivo by blocking MCL-1 antagonist-mediated cytoprotective MCL-1 accumulation by promoting degradative ubiquitination, disrupting STAT-3-mediated transcription, and inducing NOXA-mediated MCL-1 degradation. They also suggest that this strategy may improve MCL-1 antagonist efficacy in AML and potentially other malignancies.

Summary: Mechanisms underlying potentiation of the anti‐myeloma (MM) activity of ataxia telangiectasia Rad3 (ATR) antagonists by MAPK (Mitogen‐activated protein kinases)‐related extracellular kinase 1/2 (MEK1/2) inhibitors were investigated. Co‐administration of the ATR inhibitor (ATRi) BAY1895344 (BAY) and MEK1/2 inhibitors, for example, cobimetinib, synergistically increased cell death in diverse MM cell lines. Mechanistically, BAY and cobimetinib blocked STAT3 Tyr705 and Ser727 phosphorylation, respectively, and dual dephosphorylation triggered marked STAT3 inactivation and downregulation of STAT3 (Signal transducer and activator of transcription 3) downstream targets (c‐Myc and BCL‐X _L ). Similar events occurred in highly bortezomib‐resistant (PS‐R) cells, in the presence of patient‐derived conditioned medium, and with alternative ATR (e.g. M1774) and MEK1/2 (trametinib) inhibitors. Notably, constitutively active STAT3 c‐MYC or BCL‐X _L ectopic expression significantly protected cells from BAY/cobimetinib. In contrast, transfection of cells with a dominant‐negative form of STAT3 (Y705F) sensitized cells to cobimetinib, as did ATR shRNA knockdown. Conversely, MEK1/2 knockdown markedly increased ATRi sensitivity. The BAY/cobimetinib regimen was also active against primary CD138 ⁺ MM cells, but not normal CD34 ⁺ cells. Finally, the ATR inhibitor/cobimetinib regimen significantly improved survival in MM xenografts, including bortezomib‐resistant models, with minimal toxicity. Collectively, these findings suggest that combined ATR/MEK1/2 inhibition triggers dual STAT3 Tyr705 and Ser727 dephosphorylation, pronounced downregulation of cytoprotective targets and MM cell death, warranting attention as a novel therapeutic strategy in MM.

The characterization of the molecular landscape and the advent of targeted therapies have defined a new era in the prognostication and treatment of acute myeloid leukemia. Recent revisions in the European LeukemiaNet 2022 guidelines have refined the molecular, cytogenetic, and treatment-related boundaries between myelodysplastic neoplasms (MDS) and AML. This review details the molecular mechanisms and cellular pathways of myeloid maturation aberrancies contributing to dysplasia and leukemogenesis, focusing on recent molecular categories introduced in ELN 2022. We provide insights into novel and rational therapeutic combination strategies that exploit mechanisms of leukemogenesis, highlighting the underpinnings of splicing factors, the cohesin complex, and chromatin remodeling. Areas of interest for future research are summarized, and we emphasize approaches designed to advance existing treatment strategies.

Purpose: The goal of this study was to characterize the relationship between ATR and STAT3 interactions in human multiple myeloma (MM) cells. Methods: Various MM cell lines, including IL-6-dependent cells were exposed to ATR inhibitors and effects on STAT3 Tyr705 and Ser727 were monitored by WB analysis and ImageStream analysis. Parallel studies examined induction of cell death, STAT3 DNA binding activity, and expression of STAT3 downstream targets (BCL-X_L, MCL-1, c-MYC). Validation was obtained in ATR shRNA knock-down cells, and in cells ectopically expressing BCL-X_L, MCL-1, or c-MYC. Analogous studies were performed in primary MM cells and in a MM xenograft model. Results: Multiple pharmacologic ATR inhibitors inhibited STAT3 Tyr705 (but not Ser727) phosphorylation at low uM concentrations and down-regulated BCL-X_L, MCL-1, c-MYC in association with cell death induction. Compatible results were observed in ATR shRNA knock-down cells. Cell death induced by ATR inhibitors was significantly attenuated in cells ectopically expressing constitutively active STAT3, BCL-X_L, MCL-1, or c-MYC. Concordant results were observed in primary human MM cells and in an in vivo MM xenograft model. Conclusions: Collectively, these findings argue for a non-canonical role for the ATR kinase in STAT3 activation in MM cells, and suggest that STAT3 inactivation contributes to the lethal actions of ATR inhibitors in MM.

The relationship between the checkpoint kinase Chk1 and the STAT3 pathway was examined in multiple myeloma cells. Gene expression profiling of U266 cells exposed to low (nmol/L) Chk1 inhibitor [PF-477736 (PF)] concentrations revealed STAT3 pathway-related gene downregulation (e.g., BCL-XL, MCL-1, c-Myc), findings confirmed by RT-PCR. This was associated with marked inhibition of STAT3 Tyr705 (but not Ser727) phosphorylation, dimerization, nuclear localization, DNA binding, STAT3 promoter activity by chromatin immunoprecipitation assay, and downregulation of STAT-3-dependent proteins. Similar findings were obtained in other multiple myeloma cells and with alternative Chk1 inhibitors (e.g., prexasertib, CEP3891). While PF did not reduce GP130 expression or modify SOCS or PRL-3 phosphorylation, the phosphatase inhibitor pervanadate antagonized PF-mediated Tyr705 dephosphorylation. Significantly, PF attenuated Chk1-mediated STAT3 phosphorylation in in vitro assays. Surface plasmon resonance analysis suggested Chk1/STAT3 interactions and PF reduced Chk1/STAT3 co-immunoprecipitation. Chk1 CRISPR knockout or short hairpin RNA knockdown cells also displayed STAT3 inactivation and STAT3-dependent protein downregulation. Constitutively active STAT3 diminished PF-mediated STAT3 inactivation and downregulate STAT3-dependent proteins while significantly reducing PF-induced DNA damage (γH2A.X formation) and apoptosis. Exposure of cells with low basal phospho-STAT3 expression to IL6 or human stromal cell conditioned medium activated STAT3, an event attenuated by Chk1 inhibitors. PF also inactivated STAT3 in primary human CD138+ multiple myeloma cells and tumors extracted from an NSG multiple myeloma xenograft model while inhibiting tumor growth.Implications:These findings identify a heretofore unrecognized link between the Chk1 and STAT3 pathways and suggest that Chk1 pathway inhibitors warrant attention as novel and potent candidate STAT3 antagonists in myeloma.

The relationship between the checkpoint kinase Chk1 and the STAT3 pathway was examined in multiple myeloma cells. Gene expression profiling of U266 cells exposed to low (nmol/L) Chk1 inhibitor [PF-477736 (PF)] concentrations revealed STAT3 pathway-related gene downregulation (e.g., BCL-XL, MCL-1, c-Myc), findings confirmed by RT-PCR. This was associated with marked inhibition of STAT3 Tyr705 (but not Ser727) phosphorylation, dimerization, nuclear localization, DNA binding, STAT3 promoter activity by chromatin immunoprecipitation assay, and downregulation of STAT-3-dependent proteins. Similar findings were obtained in other multiple myeloma cells and with alternative Chk1 inhibitors (e.g., prexasertib, CEP3891). While PF did not reduce GP130 expression or modify SOCS or PRL-3 phosphorylation, the phosphatase inhibitor pervanadate antagonized PF-mediated Tyr705 dephosphorylation. Significantly, PF attenuated Chk1-mediated STAT3 phosphorylation in in vitro assays. Surface plasmon resonance analysis suggested Chk1/STAT3 interactions and PF reduced Chk1/STAT3 co-immunoprecipitation. Chk1 CRISPR knockout or short hairpin RNA knockdown cells also displayed STAT3 inactivation and STAT3-dependent protein downregulation. Constitutively active STAT3 diminished PF-mediated STAT3 inactivation and downregulate STAT3-dependent proteins while significantly reducing PF-induced DNA damage (γH2A.X formation) and apoptosis. Exposure of cells with low basal phospho-STAT3 expression to IL6 or human stromal cell conditioned medium activated STAT3, an event attenuated by Chk1 inhibitors. PF also inactivated STAT3 in primary human CD138+ multiple myeloma cells and tumors extracted from an NSG multiple myeloma xenograft model while inhibiting tumor growth.Implications:These findings identify a heretofore unrecognized link between the Chk1 and STAT3 pathways and suggest that Chk1 pathway inhibitors warrant attention as novel and potent candidate STAT3 antagonists in myeloma.