Small Molecule Multi-Targeted Kinase Inhibitor RGB-286638 Triggers P53-Dependent and -Independent Anti-Multiple Myeloma Activity through Inhibition of Transcriptional CDKs

Small molecule multi-targeted CDK inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638’s mode-of-action in MM cell lines with wild type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638-triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA binding activity. Additionally, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1, and miR-21. Our data provide the rationale for the development of transcriptional CDK inhibitors as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells

The KDM3A-KLF2-IRF4 axis maintains myeloma cell survival

KDM3A is implicated in tumorigenesis; however, its biological role in multiple myeloma (MM) has not been elucidated. Here we identify KDM3A-KLF2-IRF4 axis dependence in MM. Knockdown of KDM3A is toxic to MM cells in vitro and in vivo. KDM3A maintains expression of KLF2 and IRF4 through H3K9 demethylation, and knockdown of KLF2 triggers apoptosis. Moreover, KLF2 directly activates IRF4 and IRF4 reciprocally upregulates KLF2, forming a positive autoregulatory circuit. The interaction of MM cells with bone marrow milieu mediates survival of MM cells. Importantly, silencing of KDM3A, KLF2 or IRF4 both decreases MM cell adhesion to bone marrow stromal cells and reduces MM cell homing to the bone marrow, in association with decreased ITGB7 expression in MAF-translocated MM cell lines. Our results indicate that the KDM3A-KLF2-IRF4 pathway plays an essential role in MM cell survival and homing to the bone marrow, and therefore represents a therapeutic target

Combination of a Selective HSP90α/β Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells

Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM

A proto-oncogene BCL6 is up-regulated in the bone marrow microenvironment in multiple myeloma cells

Constitutive B-cell lymphoma 6 (Bcl-6) expression was undetectable in multiple myeloma (MM) cell lines, except U266 cells. However, it was up-regulated by coculture with bone marrow (BM) stromal cell-culture supernatant (SCCS). Bcl-6 expression in patient MM cells in the BM was positive. Anti–interleukin-6 (IL-6)–neutralizing antibody significantly blocked SCCS-induced Bcl-6 in MM cells. Indeed, IL-6 strongly triggered Bcl-6 expression in MM cells, whereas Janus kinase inhibitor and STAT3 siRNA down-regulated Bcl-6. Tumor necrosis factor-α (TNF-α) also triggered Bcl-6, but independently of STAT3, whereas IκB kinaseβ inhibitor down-regulated TNF-α–induced Bcl-6, indicating that the canonical nuclear factor-κB pathway mediates TNF-α–induced Bcl-6 expression. Importantly, down-regulation of Bcl-6 by shRNA significantly inhibited MM cell growth in the presence of SCCS. Our results therefore suggest that Bcl-6 expression in MM cells is modulated, at least in part, via Janus kinase/STAT3 and canonical nuclear factor-κB pathways and that targeting Bcl-6, either directly or via these cascades, inhibits MM cell growth in the BM milieu