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BH3-mimetics targeting anti-apoptotic proteins such as MCL-1 (S63845) or BCL-2 (venetoclax) are currently being evaluated as effective therapies for the treatment of multiple myeloma (MM). Interleukin 6, produced by mesenchymal stromal cells (MSCs), has been shown to modify the expression of anti-apoptotic proteins and their interaction with the pro-apoptotic BIM protein in MM cells. In this study, we assess the efficacy of S63845 and venetoclax in MM cells in direct co-culture with MSCs derived from MM patients (pMSCs) to identify additional mechanisms involved in the stroma-induced resistance to these agents. MicroRNAs miR-193b-3p and miR-21-5p emerged among the top deregulated miRNAs in myeloma cells when directly co-cultured with pMSCs, and we show their contribution to changes in MCL-1 and BCL-2 protein expression and in the activity of S63845 and venetoclax. Additionally, direct contact with pMSCs under S63845 and/or venetoclax treatment modifies myeloma cell dependence on different BCL-2 family anti-apoptotic proteins in relation to BIM, making myeloma cells more dependent on the non-targeted anti-apoptotic protein or BCL-XL. Finally, we show a potent effect of the combination of S63845 and venetoclax even in the presence of pMSCs, which supports this combinatorial approach for the treatment of MM.

Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD.

[Background]: Proviral Insertion site for Moloney murine leukemia virus (PIM) kinases are overexpressed in hematologic malignancies, including multiple myeloma. Previous preclinical data from our group demonstrated the anti-myeloma effect of the pan-PIM kinase inhibitor PIM447. [Methods]: Based on those data, we evaluate here, by in vitro and in vivo studies, the activity of the triple combination of PIM447 + pomalidomide + dexamethasone (PIM-Pd) in multiple myeloma. [Results]: Our results show that the PIM-Pd combination exerts a potent anti-myeloma effect in vitro and in vivo, where it markedly delays tumor growth and prolongs survival of treated mice. Mechanism of action studies performed in vitro and on mice tumor samples suggest that the combination PIM-Pd inhibits protein translation processes through the convergent inhibition of c-Myc and mTORC1, which subsequently disrupts the function of eIF4E. Interestingly the MM pro-survival factor IRF4 is also downregulated after PIM-Pd treatment. As a whole, all these molecular changes would promote cell cycle arrest and deregulation of metabolic pathways, including glycolysis and lipid biosynthesis, leading to inhibition of myeloma cell proliferation. [Conclusions]: Altogether, our data support the clinical evaluation of the triple combination PIM-Pd for the treatment of patients with multiple myeloma.

The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.

[Purpose]: The search for new drugs that control the continuous relapses of multiple myeloma is still required. Here, we report for the first time the potent antimyeloma activity of amiloride, an old potassium-sparing diuretic approved for the treatment of hypertension and edema due to heart failure. [Experimental Design]: Myeloma cell lines and primary samples were used to evaluate cytotoxicity of amiloride. In vivo studies were carried out in a xenograft mouse model. The mechanisms of action were investigated using RNA-Seq experiments, qRT-PCR, immunoblotting, and immunofluorescence assays. [Results]: Amiloride-induced apoptosis was observed in a broad panel of multiple myeloma cell lines and in a xenograft mouse model. Moreover, amiloride also had a synergistic effect when combined with dexamethasone, melphalan, lenalidomide, and pomalidomide. RNA-Seq experiments showed that amiloride not only significantly altered the level of transcript isoforms and alternative splicing events, but also deregulated the spliceosomal machinery. In addition, disruption of the splicing machinery in immunofluorescence studies was associated with the inhibition of myeloma cell viability after amiloride exposure. Although amiloride was able to induce apoptosis in myeloma cells lacking p53 expression, activation of p53 signaling was observed in wild-type and mutated TP53 cells after amiloride exposure. On the other hand, we did not find a significant systemic toxicity in mice treated with amiloride. [Conclusions]: Overall, our results demonstrate the antimyeloma activity of amiloride and provide a mechanistic rationale for its use as an alternative treatment option for relapsed multiple myeloma patients, especially those with 17p deletion or TP53 mutations that are resistant to current therapies.

[Purpose]: PIM kinases are a family of serine/threonine kinases recently proposed as therapeutic targets in oncology. In the present work, we have investigated the effects of the novel pan-PIM kinase inhibitor, PIM447, on myeloma cells and myeloma-associated bone disease using different preclinical models. [Experimental Design]: In vitro/ex vivo cytotoxicity of PIM447 was evaluated on myeloma cell lines and patient samples. Synergistic combinations with standard treatments were analyzed with Calcusyn Software. PIM447 effects on bone cells were assessed on osteogenic and osteoclastogenic cultures. The mechanisms of PIM447 were explored by immunoblotting, qPCR, and immunofluorescence. A murine model of disseminated multiple myeloma was employed for in vivo studies. [Results]: PIM447 is cytotoxic for myeloma cells due to cell-cycle disruption and induction of apoptosis mediated by a decrease in phospho-Bad (Ser112) and c-Myc levels and the inhibition of mTORC1 pathway. Importantly, PIM447 demonstrates a very strong synergy with different standard treatments such as bortezomib + dexamethasone (combination index, CI = 0.002), lenalidomide + dexamethasone (CI = 0.065), and pomalidomide + dexamethasone (CI = 0.077). PIM447 also inhibits in vitro osteoclast formation and resorption, downregulates key molecules involved in these processes, and partially disrupts the F-actin ring, while increasing osteoblast activity and mineralization. Finally, PIM447 significantly reduced the tumor burden and prevented tumor-associated bone loss in a disseminated murine model of human myeloma. [Conclusions]: Our results demonstrate dual antitumoral and bone-protective effects of PIM447. This fact, together with the very strong synergy exhibited with standard-of-care treatments, supports the future clinical development of this drug in multiple myeloma.

Despite new advances in multiple myeloma treatment and the consequent improvement in overall survival, most patients relapse or become refractory to treatment. This suggests that new molecules and combinations that may further inhibit important survival pathways for these tumor cells are needed. In this context, zalypsis is a novel compound, derived from marine organisms, with a powerful preclinical anti-myeloma effect based on the sensitivity of malignant plasma cells to DNA-damage induction; and it has already been tested in a phase I/II clinical trial in multiple myeloma. We hypothesized that the addition of this compound to the combination of bortezomib plus dexamethasone may improve efficacy with acceptable toxicity. The triple combination demonstrated strong synergy and higher efficacy compared with double combinations; not only in vitro, but also ex vivo and, especially, in in vivo experiments. The triple combination triggers cell death, mainly through a synergistic induction of DNA damage and a decrease in the nuclear localization of nuclear factor kappa B. Our findings support the clinical evaluation of this combination for relapsed and refractory myeloma patients.

In the present study, Xenopus laevis embryos were exposed to a range of perfluorooctane sulfonate (PFOS) concentrations (0, 0.5, 6, 12, 24, 48 and 96 mg/L) for 96 h in laboratorial conditions to establish toxicity along with possible gene expression changes. Mortality and deformities were monitored daily and head-tail length was measured at the end of the assay as an indicator of growth. At 24 and 96 h post-exposure (hpe), the mRNA expression levels of the genetic markers involved in general stress responses (hsp70, hsp47, crh-a and ucn1), oxidative stress (cat.2 and sod), lipid metabolism (ppard) and apoptosis (tp53 and bax) were analyzed by RT-qPCR. Malformations were significantly higher in the embryos exposed to the highest PFOS concentration (41.8% to 56.4%) compared to controls (5.5%) at 48, 72 and 96 hpe. Growth inhibition was observed in the embryos exposed to PFOS concentrations≥48 mg/L. At 24 hpe, a statistically significant up-regulation of genes hsp70, hsp47, ppard, tp53 and bax in relation to controls was found. Similar responses were found for genes hsp70, hsp47, crh-a, ucn1, sod and ppard at 96 hpe. Alterations in the mRNA expression levels indicated both a stress response to PFOS exposure during X. laevis embryo development, and alterations in the regulation of oxidative stress, apoptosis, and differentiation. These molecular alterations were detected at an earlier exposure time or at lower concentrations than those producing developmental toxicity. Therefore, these sensitive warning signals could be used together with other biomarkers to supplement alternative methods (i.e. the frog embryo test) for developmental toxicity safety evaluations, and as tools in amphibian risk assessments for PFOS and its potential substitutes. © 2016 Elsevier Inc.

Knowledge about clonal diversity and selection is critical to understand multiple myeloma (MM) pathogenesis, chemoresistance and progression. If targeted therapy becomes reality, identification and monitoring of intraclonal plasma cell (PC) heterogeneity would become increasingly demanded. Here we investigated the kinetics of intraclonal heterogeneity among 116 MM patients using 23-marker multidimensional flow cytometry (MFC) and principal component analysis, at diagnosis and during minimal residual disease (MRD) monitoring. Distinct phenotypic subclones were observed in 35116 (30%) newly diagnosed MM patients. In 1035 patients, persistent MRD was detected after 9 induction cycles, and longitudinal comparison of patient-paired diagnostic vs MRD samples unraveled phenotypic clonal tiding after therapy in half (510) of the patients. After demonstrating selection of distinct phenotypic subsets by therapeutic pressure, we investigated whether distinct fluorescence-activated cell-sorted PC subclones had different clonogenic and cytogenetic profiles. In half (510) of the patients analyzed, distinct phenotypic subclones showed different clonogenic potential when co-cultured with stromal cells, and in 611 cases distinct phenotypic subclones displayed unique cytogenetic profiles by interphase fluorescence in situ hybridization, including selective del(17p13). Collectively, we unravel potential therapeutic selection of preexisting diagnostic phenotypic subclones during MRD monitoring; because phenotypically distinct PCs may show different clonogenic and cytogenetic profiles, identification and follow-up of unique phenotypic-genetic myeloma PC subclones may become relevant for tailored therapy.

Despite remarkable therapeutic improvement in multiple myeloma (MM), prognosis is very poor once patients become refractory to or ineligible for proteasome inhibitors and immunomodulatory drugs. Accordingly, new drugs are clearly needed for this patient population, and extensive efforts are leading to a new breed of antimyeloma drugs.

The development of resistance to therapy is unavoidable in the history of multiple myeloma patients. Therefore, the study of its characteristics and mechanisms is critical in the search for novel therapeutic approaches to overcome it. This effort is hampered by the absence of appropriate preclinical models, especially those mimicking acquired resistance. Here we present an in vivo model of acquired resistance based on the continuous treatment of mice bearing subcutaneous MM1S plasmacytomas. Xenografts acquired resistance to two generations of immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide) in combination with dexamethasone, that was reversible after a wash-out period. Furthermore, lenalidomide-dexamethasone (LD) or pomalidomide-dexamethasone (PD) did not display cross-resistance, which could be due to the differential requirements of the key target Cereblon and its substrates Aiolos and Ikaros observed in cells resistant to each combination. Differential gene expression profiles of LD and PD could also explain the absence of cross-resistance. Onset of resistance to both combinations was accompanied by upregulation of the mitogen-activated protein kinaseextracellular signal-regulated kinase (ERK) kinase (MEK)ERK pathway and addition of selumetinib, a small-molecule MEK inhibitor, could resensitize resistant cells. Our results provide insights into the mechanisms of acquired resistance to LD and PD combinations and offer possible therapeutic approaches to addressing IMiD resistance in the clinic.

The regulation of hematopoietic stem cells (HSCs) depends on the integration of the multiple signals received from the bone marrow niche. We show the relevance of the protein tyrosine phosphatase PTPN13 and b-catenin as intracellular signaling molecules to control HSCs adhesiveness, cell cycling, and quiescence. Lethally irradiated mice transplanted with Lin– bone marrow cells in which PTPN13 or b-catenin had been silenced showed a significant increase of long-term (LT) and short-term (ST) HSCs. A decrease in cycling cells was also found, together with an increase in quiescence. The decreased expression of PTPN13 or b-catenin was linked to the upregulation of several genes coding for integrins and several cadherins, explaining the higher cell adhesiveness. Our data are consistent with the notion that the levels of PTPN13 and b-catenin must be strictly regulated by extracellular signaling to regulate HSC attachment to the niche and the balance between proliferation and quiescence.