Anti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the ...soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup- and IL-6-induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup-induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity.
Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of ...the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.
Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent ...observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD+-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM.
•SIRT6 is highly expressed in multiple myeloma cells and blocks expression of ERK-regulated genes.•Targeting SIRT6 enzymatic activity sensitizes multiple myeloma cells to DNA-damaging agents.
There is growing evidence that spleen tyrosine kinase (SYK) is critical for acute myeloid leukaemia (AML) transformation and maintenance of the leukemic clone in AML patients. It has also been found ...to be over-expressed in AML patients, with activating mutations in foetal liver tyrosine kinase 3 (FLT3), particularly those with internal tandem duplications (FLT3-ITD), where it transactivates FLT3-ITD and confers resistance to treatment with FLT3 tyrosine kinase inhibitors (TKIs).
We have previously described a pharmacological approach to treating FLT3-ITD-positive AML that relies on proteasome-mediated FLT3 degradation via inhibition of USP10, the deubiquitinating enzyme (DUB) responsible for cleaving ubiquitin from FLT3.
Here, we show that USP10 is also a major DUB required for stabilisation of SYK. We further demonstrate that degradation of SYK can be induced by USP10-targeting inhibitors. USP10 inhibition leads to death of cells driven by active SYK or oncogenic FLT3 and potentiates the anti-leukemic effects of FLT3 inhibition in these cells.
We suggest that USP10 inhibition is a novel approach to inhibiting SYK and impeding its role in the pathology of AML, including oncogenic FLT3-positive AML. Also, given the significant transforming role SYK in other tumours, targeting USP10 may have broader applications in cancer.
Oncogenic activated RAS mutations have been detected in 50% of de novo and 70% of relapsed multiple myeloma (MM) patients. Translocation t(11;14) involving IgH/CCDN1 and overexpression of cyclin-Ds ...are early events in MM pathogenesis, enhancing uncontrolled MM cell growth. We hypothesized that targeting both RAS/MAPK pathway molecules including Erk1/2 along with cyclin-Ds enhances MM cytotoxicity and minimizes side effects. Recent studies have demonstrated the high potency of Erk1/2 and CDK4/6 inhibitors in metastatic relapsed cancers, and here we tested anti-MM effects of the Erk1/2 + CDK4/6 inhibitor combination. Our studies showed strong synergistic (IC < 0.5) cytotoxicity of Erk1/2i + CDK4/6i in MM-cells. Erk1/2i + CDK4/6i treatment in a dose-dependent manner arrested MM-cells in the G0/G1 phase and activated mitochondrial apoptotic signaling. Our studies showed that Erk1/2i + CDK4/6i treatment-induced inhibition of key target molecules in Erk1/2 and CDK4/6 signaling, such as c-myc, p-RSK, p-S6, p-RB, and E2F1, suggesting on-target activity of these inhibitors. We identified Erk1/2i + CDK4/6i treatment associated five-gene signature which includes SNRPB and SLC25A5; these genes are involved in RNA processing and mitochondrial metabolism, respectively. Overall, our studies provide the preclinical framework for Erk1/2i + CDK4/6i combination clinical trials to target Ras+CDK pathways to improve patient outcome in MM.
Over the last few years, a detailed map of genetic and epigenetic lesions that underlie multiple myeloma (MM) has been created. Regulation of microRNA (miR)-dependent gene expression and mRNA ...splicing play significant roles in MM pathogenesis; however, to date an interplay between these processes is not yet delineated. Here we investigated miR-mediated regulation of splicing networks at the transcriptome level. Our studies show that a significant number (78%) of miRs which are either up- or down-regulated in patient CD138+ MM cells, but not in healthy donors (HD) CD138+ plasma cells (PC), target genes involved in early stages of pre-mRNA splicing. We also identified deregulated miRs that target core splicing factors (SF) and modifiers (SM, enhancers/silencers) which cause altered splicing in MM. Our studies suggest that Let-7f, in combination other miRs which are frequently and significantly deregulated in patients with overt MM, targets genes that regulate intron excision. Importantly, deregulated expression of certain miRs in MM promote increased intron retention, a novel characteristic of the MM genome, by inducing deregulated expression of the genes that regulate the splicing network. Our studies, therefore, provide the rationale for therapeutically targeting deregulated miRs to reverse aberrant splicing and improve patient outcome in MM.
Recurrence of multiple myeloma (MM) after therapy suggests the presence of tumor-initiating subpopulations. In our study, we performed flow cytometry–based Hoechst 33342 staining to evaluate the ...existence of a MM population with stem-like features known as side population (SP) cells. SP cells exhibit substantial heterogeneity in MM cell lines and primary MM cells; express CD138 antigen in MM cell lines; display higher mRNA expression and functional activity of ABCG2 transporter; and have a higher proliferation index compared with non-SP cells. We observed evidence for clonogenic potential of SP cells, as well as the ability of SP cells to regenerate original population. Moreover, SP cells revealed higher tumorigenicity compared with non-SP cells. Importantly, lenalidomide decreased the percentage and clonogenicity of SP cells, and also induced phosphorylation changes in Akt, GSK-3α/β, MEK1, c-Jun, p53, and p70S6K in SP cells. Adherence to bone marrow stromal cells (BMSCs) increased the percentage, viability, and proliferation potential of SP cells. Lenalidomide and thalidomide abrogated this stimulatory effect of BMSCs and significantly decreased the percentage of SP cells. Our studies demonstrate a novel mechanism of action for lenalidomide, namely targeting SP fraction, providing the framework for new therapeutic strategies targeting subpopulations of MM cells including presumptive stem cells.
Nucleoside analogs (NAs) are considered as appropriate agents in the treatment of Waldenström macroglobulinemia (WM), a lymphoplasmacytic lymphoma. Sporadic reports on increased incidence of ...transformation to high-grade non-Hodgkin's lymphoma and development of therapy-related myelodysplasia/acute leukemia (t-MDS/AML) among patients with WM treated with NAs prompted us to examine the incidence of such events in a large population of patients with WM.
We examined the incidence of these events in 439 patients with WM, 193 and 136 of whom were previously treated with and without an NA, respectively, and 110 of whom had similar long-term follow-up without treatment. The median follow-up for all patients was 5 years.
Overall, 12 patients (6.2%) either developed transformation (n = 9; 4.7%) or developed t-MDS/AML (n = 3; 1.6%) among NA-treated patients, compared with one patient (0.4%) who developed transformation in the non-NA treated group (P < .001); no such events occurred among untreated patients. Transformation and t-MDS/AML occurred at a median of 5 years from onset of NA therapy. The median survival of NA-treated patients who developed transformation did not differ from other NA-treated patients as a result of effective salvage treatment used for transformed disease. However, all NA-treated patients who developed t-MDS/AML died at a median of 5 months.
These data demonstrate an increased incidence of disease transformation to high-grade NHL and the development of t-MDS/AML among patients with WM treated with NAs.
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