THZ531 Induces a State of BRCAness in Multiple Myeloma Cells: Synthetic Lethality with Combination Treatment of THZ 531 with DNA Repair Inhibitors

Multiple myeloma (MM) is a hematological disease marked by abnormal growth of B cells in bone marrow. Inherent chromosomal instability and DNA damage are major hallmarks of MM, which implicates an aberrant DNA repair mechanism. Studies have implicated a role for CDK12 in the control of expression of DNA damage response genes. In this study, we examined the effect of a small molecule inhibitor of CDK12–THZ531 on MM cells. Treatment of MM cells with THZ531 led to heightened cell death accompanied by an extensive effect on gene expression changes. In particular, we observed downregulation of genes involved in DNA repair pathways. With this insight, we extended our study to identify synthetic lethal mechanisms that could be exploited for the treatment of MM cells. Combination of THZ531 with either DNA-PK inhibitor (KU-0060648) or PARP inhibitor (Olaparib) led to synergistic cell death. In addition, combination treatment of THZ531 with Olaparib significantly reduced tumor burden in animal models. Our findings suggest that using a CDK12 inhibitor in combination with other DNA repair inhibitors may establish an effective therapeutic regimen to benefit myeloma patients

THZ531 Induces a State of BRCAness in Multiple Myeloma Cells: Synthetic Lethality with Combination Treatment of THZ 531 with DNA Repair Inhibitors

10.3390/ijms23031207INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES23

Genome Wide Expression Profiling of Cancer Cell Lines Cultured in Microgravity Reveals Significant Dysregulation of Cell Cycle and MicroRNA Gene Networks

<div><p>Zero gravity causes several changes in metabolic and functional aspects of the human body and experiments in space flight have demonstrated alterations in cancer growth and progression. This study reports the genome wide expression profiling of a colorectal cancer cell line-DLD-1, and a lymphoblast leukemic cell line-MOLT-4, under simulated microgravity in an effort to understand central processes and cellular functions that are dysregulated among both cell lines. Altered cell morphology, reduced cell viability and an aberrant cell cycle profile in comparison to their static controls were observed in both cell lines under microgravity. The process of cell cycle in DLD-1 cells was markedly affected with reduced viability, reduced colony forming ability, an apoptotic population and dysregulation of cell cycle genes, oncogenes, and cancer progression and prognostic markers. DNA microarray analysis revealed 1801 (upregulated) and 2542 (downregulated) genes (>2 fold) in DLD-1 cultures under microgravity while MOLT-4 cultures differentially expressed 349 (upregulated) and 444 (downregulated) genes (>2 fold) under microgravity. The loss in cell proliferative capacity was corroborated with the downregulation of the cell cycle process as demonstrated by functional clustering of DNA microarray data using gene ontology terms. The genome wide expression profile also showed significant dysregulation of post transcriptional gene silencing machinery and multiple microRNA host genes that are potential tumor suppressors and proto-oncogenes including <i>MIR22HG</i>, <i>MIR17HG</i> and <i>MIR21HG</i>. The <i>MIR22HG</i>, a tumor-suppressor gene was one of the highest upregulated genes in the microarray data showing a 4.4 log fold upregulation under microgravity. Real time PCR validated the dysregulation in the host gene by demonstrating a 4.18 log fold upregulation of the miR-22 microRNA. Microarray data also showed dysregulation of direct targets of miR-22, <i>SP1</i>, <i>CDK6</i> and <i>CCNA2</i>.</p></div

Chromatin remodeling mediated by ARID1A is indispensable for normal hematopoiesis in mice.

Precise regulation of chromatin architecture is vital to physiological processes including hematopoiesis. ARID1A is a core component of the mammalian SWI/SNF complex, which is one of the ATP-dependent chromatin remodeling complexes. To uncover the role of ARID1A in hematopoietic development, we utilized hematopoietic cell-specific deletion of Arid1a in mice. We demonstrate that ARID1A is essential for maintaining the frequency and function of hematopoietic stem cells and its loss impairs the differentiation of both myeloid and lymphoid lineages. ARID1A deficiency led to a global reduction in open chromatin and ensuing transcriptional changes affected key genes involved in hematopoietic development. We also observed that silencing of ARID1A affected ATRA-induced differentiation of NB4 cells, suggesting its role in granulocytic differentiation of human leukemic cells. Overall, our study provides a comprehensive elucidation of the function of ARID1A in hematopoiesis and highlights the central role of ARID1A-containing SWI/SNF complex in maintaining chromatin dynamics in hematopoietic cells

Functional annotation of microarray data using DAVID.

<p><b>A</b> Functional annotation of upregulated and downregulated genes of DLD-1 cells under microgravity <b>B</b> Functional annotation of upregulated and downregulated genes of MOLT-4 cells under microgravity.</p

Effect of microgravity on cell morphology and cell viability of DLD-1 cell cultures.

<p><b>A</b> DLD-1 cell cultures; Static culture (control) <b>B</b> DLD-1 Microgravity culture at 16 RPM <b>C</b> DLD-1 Microgravity culture at 27RPM <b>Differential staining to detect apoptotic population D</b> DLD-1Static monolayer cultures <b>E</b> Microgravity cultures of DLD1 at 16 RPM <b>F</b> Microgravity cultures of DLD1 at 27 RPM <b>G Cell adhesion and proliferation assay</b> Top panel—static cultures, bottom panel—microgravity cultures shifted to static TCP <b>H</b> Morphological changes in DLD-1; Crystal violet staining of DLD-1 cells in static monolayer culture <b>I</b> Crystal violet staining of DLD-1 cells after transfer of cell aggregates from microgravity to TCP <b>J</b> Colony forming ability assay; Static cultures <b>K</b> Colony forming ability assay; DLD-1 cells after transfer of cell aggregates from microgravity to TCP</p

Effect of microgravity on cell viability and cell cycle of DLD-1 and MOLT-4 cell lines.

<p><b>A</b> Cell viability assay for DLD-1 cells; Viability measured for microgravity cultures (16 RPM and 27 RPM) and static cultures using MTT <b>B</b> Cell cycle analysis for DLD-1 cells; Static <b>C</b> Cell cycle analysis; Microgravity <b>D</b> Cell cycle analysis; Static suspensions on agar underlays <b>E</b> The average sub G0 population in replicates of cell cycle analysis for microgravity, static and static suspension cultures of DLD-1 cells <b>F MOLT-4 cell culture</b> Static and Microgravity cultures of MOLT-4 <b>G Cell viability assay</b> Viability measured for microgravity cultures (16 RPM and 27 RPM) and static cultures using MTT <b>H</b> Cell cycle analysis; Static <b>I</b> Cell cycle analysis; Microgravity cultures of MOLT-4.</p

Analysis of microarray data using Gene Functional Classification and Functional Annotation; Dysregulation of genes involved in the Notch signaling system and microRNA processing and regulation.

<p><b>A</b> Regulation of transcription <b>B</b> RNA mediated gene silencing (PTGS) <b>C</b> Notch signaling pathway <b>D</b> Dysregulated microRNA processors and regulators.</p