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BH3 mimetics are promising drugs for hematologic malignancies that trigger cell death by promoting the release of proapoptotic BCL2 family members from antiapoptotic proteins. Multiple myeloma is considered to be a disease dependent mainly on MCL1 for survival, based mostly on studies using cell lines. We used a BH3-mimetic toolkit to study the dependency on BCL2, BCLXL, or MCL1 in malignant plasma cells from 60 patients. Dependencies were analyzed using an unbiased BH3 mimetics cell-death clustering by k-means. In the whole cohort of patients, BCL2 dependency was mostly found in the CCND1 subgroup (83%). Of note, MCL1 dependence significantly increased from 33% at diagnosis to 69% at relapse, suggesting a plasticity of the cellular dependency favoring MCL1 dependencies at relapse. In addition, 35% of overall patient samples showed codependencies on either BCL2/MCL1 or BCLXL/MCL1. Finally, we identified a group of patients not targeted by any of the BH3 mimetics, predominantly at diagnosis in patients not presenting the common recurrent translocations. Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection from cell death observed by BAK knock-down, as well as the complete and early disruption of MCL1/BAK complexes on A1210477 treatment. Interestingly, this complex was also dissociated in A1210477-resistant cells, but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. In conclusion, our study opens the way to rationally use venetoclax and/or MCL1 BH3 mimetics for clinical evaluation in myeloma at both diagnosis and relapse., (© 2018 by The American Society of Hematology.)

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma associated with poor prognosis, and despite recent improvements in the therapeutic strategies for treating MCL, its management remains challenging. While improvements in next generation sequencing technology have greatly increased our understanding of the intrinsic abnormalities of MCL, the role of extrinsic signaling remains largely unknown. Recent studies have highlighted the central role of the MCL microenvironment in tumor cell survival, drug resistance and proliferation. Characterization of the diverse MCL tumoral niches and comprehension of the crosstalk between tumor cells and surrounding cells within the MCL microenvironment are needed to increase treatment efficacy. Here, we reviewed the recent findings regarding the MCL microenvironment that could be rapidly translated into new therapeutic strategies to overcome drug resistance during MCL treatment.

The aggressive biological behavior of mantle cell lymphoma (MCL) and its short response to current treatment highlight a great need for better rational therapy. Herein, we investigate the ability of ABT-199, the Bcl-2-selective BH3 mimetic, to kill MCL cells. Among MCL cell lines tested (n = 8), only three were sensitive (LD50 < 200 nM). In contrast, all primary MCL samples tested (n = 11) were highly sensitive to ABT-199 (LD50 < 10 nM). Mcl-1 and Bcl-xL both confer resistance to ABT-199-specific killing and BCL2/(BCLXL+MCL1) mRNA ratio is a strong predictor of sensitivity. By mimicking the microenvironment through CD40 stimulation, we show that ABT-199 sensitivity is impaired through activation of NF-kB pathway and Bcl-x(L) up-regulation. We further demonstrate that resistance is rapidly lost when MCL cells detach from CD40L-expressing fibroblasts. It has been reported that ibrutinib induces lymphocytosis in vivo holding off malignant cells from their protective microenvironment. We show here for two patients undergoing ibrutinib therapy that mobilized MCL cells are highly sensitive to ABT-199. These results provide evidence that in situ ABT-199 resistance can be overcome when MCL cells escape from the lymph nodes. Altogether, our data support the clinical application of ABT-199 therapy both as a single agent and in sequential combination with BTK inhibitors.

The aim of this study was to assess the efficiency of p53 reactivation and induction of massive apoptosis (PRIMA-1(Met)) in inducing myeloma cell death, using 27 human myeloma cell lines (HMCLs) and 23 primary samples. Measuring the lethal dose (LD50) of HMCLs revealed that HMCLs displayed heterogeneous sensitivity, with an LD50 ranging from 4 μM to more than 200 μM. The sensitivity of HMCLs did not correlate with myeloma genomic heterogeneity or TP53 status, and PRIMA-1(Met) did not induce or increase expression of the p53 target genes CDKN1A or TNFRSF10B/DR5. However, PRIMA-1(Met) increased expression of NOXA in a p53-independent manner, and NOXA silencing decreased PRIMA1(Met)-induced cell death. PRIMA-1(Met) depleted glutathione (GSH) content and induced reactive oxygen species production. The expression of GSH synthetase correlated with PRIMA-1(Met) LD50 values, and we showed that a GSH decrease mediated by GSH synthetase silencing or by and L-buthionine sulphoximine, an irreversible inhibitor of γ-glutamylcysteine synthetase, increased PRIMA-1(Met)-induced cell death and overcame PRIMA-1(Met) resistance. PRIMA-1(Met) (10 μM) induced cell death in 65% of primary cells independent of the presence of del17p; did not increase DR5 expression, arguing against an activation of p53 pathway; and synergized with L-buthionine sulphoximine in all samples. Finally, we showed in mouse TP53(neg) JJN3-xenograft model that PRIMA-1(Met) inhibited myeloma growth and synergized with L-buthionine sulphoximine in vivo., (© 2014 by The American Society of Hematology.)

Background: The aim of this study was to evaluate the efficacy of the p53-reactivating drugs RITA and nutlin3a in killing myeloma cells., Methods: A large cohort of myeloma cell lines (n = 32) and primary cells (n = 21) was used for this study. This cohort contained cell lines with various TP53 statuses and primary cells with various incidences of deletion of chromosome 17. Apoptosis was evaluated using flow cytometry with Apo2.7 staining of the cell lines or via the loss of the myeloma-specific marker CD138 in primary cells. Apoptosis was further confirmed by the appearance of a subG1 peak and the activation of caspases 3 and 9. Activation of the p53 pathway was monitored using immunoblotting via the expression of the p53 target genes p21, Noxa, Bax and DR5. The involvement of p53 was further studied in 4 different p53-silenced cell lines., Results: Both drugs induced the apoptosis of myeloma cells. The apoptosis that was induced by RITA was not related to the TP53 status of the cell lines or the del17p status of the primary samples (p = 0.52 and p = 0.80, respectively), and RITA did not commonly increase the expression level of p53 or p53 targets (Noxa, p21, Bax or DR5) in sensitive cells. Moreover, silencing of p53 in two TP53(mutated) cell lines failed to inhibit apoptosis that was induced by RITA, which confirmed that RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, apoptosis induced by nutlin3a was directly linked to the TP53 status of the cell lines and primary samples (p < 0.001 and p = 0.034, respectively) and nutlin3a increased the level of p53 and p53 targets in a p53-dependent manner. Finally, we showed that a nutlin3a-induced DR5 increase (≥ 1.2-fold increase) was a specific and sensitive marker (p < 0.001) for a weak incidence of 17p deletion within the samples (≤ 19%)., Conclusion: These data show that RITA, in contrast to nutlin3a, effectively induced apoptosis in a subset of MM cells independently of p53. The findings and could be of interest for patients with a 17p deletion, who are resistant to current therapies.

Background: Lenalidomide is an active immunomodulatory and antiproliferative agent in multiple myeloma. However, the molecular mechanisms driving these activities are not yet fully elucidated. Therefore, we investigated the modulation of the cytokine/growth factor patterns of myeloma cells under LEN treatment., Methods: Lenalidomide effect on myeloma cell proliferation was investigated in a myeloma cell line collection (n=23) by (3)H-thymidine incorporation. Modulation of the cytokine/growth factor patterns of myeloma cells under LEN treatment was analysed by real-time quantitative PCR., Results: Lenalidomide inhibits the proliferation of two-thirds of myeloma cell lines independently of their genetic background. We demonstrated that LEN increased TNF-α and IL-8 inflammatory cytokines and insulin-like growth factor-1 (IGF-1) growth factor in both sensitive and resistant myeloma cells to LEN., Conclusion: Lenalidomide favours a uniform TNF-α and IL-8 inflammatory and IGF-1 secretory profile of myeloma cells, an observation that raises important questions for therapeutic approaches incorporating the agent.

Multiple myeloma is a plasma cell malignancy that is heterogeneous with respect to its causative molecular abnormalities and the treatment response of patients. The Bcl-2 protein family is critical for myeloma cell survival. ABT-737 is a cell-permeant compound that binds to Bcl-2 and Bcl-x(L) but not to Mcl-1. Using a myeloma cell line collection (n = 25) representative of different molecular translocations, we showed that ABT-737 effectively kills a subset of cell lines (n = 6), with a median lethal dose ranging from 7 ± 0.4 nM to 150 ± 7.5 nM. Of interest, all sensitive cell lines harbored a t(11;14). We demonstrated that ABT-737-sensitive and ABT-737-resistant cell lines could be differentiated by the BCL2/MCL1 expression ratio. A screen of a public expression database of myeloma patients indicates that the BCL2/MCL1 ratio of t(11;14) and hyperdiploid patients was significantly higher than in all other groups (P < .001). ABT-737 first induced the disruption of Bcl-2/Bax, Bcl-2/Bik, or Bcl-2/Puma complexes, followed by the disruption of Bcl-2 heterodimers with Bak and Bim. Altogether, the identification of a subset of cell lines and primary cells effectively killed by ABT-737 alone supported the evaluation of ABT-263, an orally active counterpart to ABT-737, for the treatment of t(11;14) and hyperdiploid groups of myeloma harboring a Bcl-2(high)/Mcl-1(low) profile.

Purpose: Mantle cell lymphoma (MCL) is considered to be incurable. ABT-737 is a BH3 mimetic that targets Bcl-2, which is overexpressed in MCL and implicated in drug resistance. The present work investigated the antitumor effect of ABT-737., Experimental Design: Six MCL cell lines and primary MCL cells (n = 13) were used. Sensitivity to ABT-737 was assessed, and expression levels of Bcl-2 and Mcl-1 were analyzed. Finally, ABT-737 was combined with other cytotoxic agents to promote tailored therapy., Results: MINO and GRANTA-519 cell lines were highly sensitive to ABT-737 [the median lethal dose (LD₅₀) = 20 and 80 nmol/L, respectively], whereas other cell lines were resistant. In primary MCL cells, 46% of patients' samples were sensitive to ABT-737. The analysis of protein expression levels revealed that both sensitive cell lines and primary MCL cells could be characterized by a Bcl-2(high)/Mcl-1(low) profile, whereas resistant MCL cells contained high levels of Mcl-1. ABT-737 induced a rapid disruption of both Bcl-2/Bax and Bcl-2/Bik complexes. In addition, silencing of Mcl-1 by siRNA sensitized MCL cell lines to ABT-737. Similarly, flavopiridol, which induces Mcl-1 downregulation, in combination with ABT-737 led to a synergistic anti-MCL effect in ABT-737-resistant cell lines. This synergy was also observed when ABT-737 was combined with either bortezomib or cytarabine., Conclusions: The present work shows that ABT-737 induces strong apoptosis in MCL cells expressing a Bcl-2(high)/Mcl-1(low) profile. In ABT-737-resistant MCL cells, downregulation of Mcl-1 overcomes Mcl-1-induced resistance and synergizes ABT-737 effects. Our results strongly support the use of ABT-737 according to the Bcl-2/Mcl-1 tumor cell profiles in the treatment of MCL., (©2011 AACR.)

Background: We analyzed detailed characteristics and salvage treatment in 175 follicular lymphoma patients from the FL2000 study who were in progression after first-line therapy with or without addition of rituximab to chemotherapy and interferon., Design and Methods: The impact of using autologous stem cell transplantation and/or rituximab administration at first progression was investigated, taking into account initial therapy. With a median follow up of 31 months, 3-year event free and overall survival rates after progression were 50% (95%CI 42-58%) and 72% (95%CI 64-78%), respectively., Results: The 3-year event free rate of rituximab re-treated patients (n=112) was 52% (95%CI 41-62%) versus 40% (95%CI 24-55%) for those not receiving rituximab second line (n=53) (P=0.075). There was a significant difference in 3-year overall survival between patients receiving autologous stem cell transplantation and those not: 92% (95%CI 78-97%) versus 63% (95%CI 51-72%) (P=0.0003), respectively. In multivariate analysis, both autologous stem cell transplantation and period of progression/relapse affected event free and overall survival., Conclusions: Regardless of front-line rituximab exposure, this study supports incorporating autologous stem cell transplantation in the therapeutic approach at first relapse for follicular lymphoma patients.

Background: This study was designed to evaluate the safety and efficacy of a conventional dose of yttrium-90 ((90)Y) ibritumomab tiuxetan combined with the etoposide rabinoside acytarabine melphalan (BEAM) regimen before autologous stem cell transplantation (ASCT) in chemosensitive relapsed or refractory low-grade B-cell lymphomas., Patients and Methods: From March 2005 to August 2006, 77 prospective patients were included, 69 (90%) with follicular lymphomas., Results: The last salvage chemotherapy regimen included rituximab for 74 patients and ASCT for 75 patients. Before ASCT, rates of complete response/unconfirmed response (CR/CRu) and partial response were 77% and 23%, respectively. After zevaline-BEAM (Z-BEAM), time to >1 × 10(9)/L neutrophils was 12 days (range, 9-35 days), and time to >20 × 10(9)/L platelets was 12 days (range, 3-42 days). No other significant extrahematologic toxicity was observed. Three months after ASCT, 68 patients (88%) were in CR/CRu. After a median follow-up of 28 months, 2-year event-free survival (EFS) and overall survival were 63% and 97%, respectively, but EFS for first-relapsed patients was 72%. When using patients as their own controls, 2-year EFS was superior after ASCT and compared favorably with the duration of response of last chemotherapy (62% vs. 37%, P = .007) (Point 1.10)., Conclusion: Z-BEAM appears safe and needs to be further evaluated in a randomized trial., (2011. Published by Elsevier Inc.)

Purpose: Fractionated radioimmunotherapy targeting CD22 may substantially improve responses and outcome in non-Hodgkin's lymphoma (NHL)., Patients and Methods: A multicenter trial evaluated two or three weekly infusions of yttrium-90 ((90)Y) epratuzumab tetraxetan (humanized anti-CD22 antibody) in 64 patients with relapsed/refractory NHL, including 17 patients who underwent prior autologous stem-cell transplantation (ASCT). Objective (OR) and complete responses (CR/complete response unconfirmed [CRu]), as well as progression-free survival (PFS), were determined., Results: At the maximum total (90)Y dose of 45 mCi/m(2) (1,665 MBq/m(2)), grade 3 to 4 hematologic toxicities were reversible to grade 1 in patients with less than 25% bone marrow involvement. The overall OR rate and median PFS for all 61 evaluable patients was 62% (CR/CRu, 48%) and 9.5 months, respectively. Patients without prior ASCT obtained high OR rates of 71% (CR/CRu, 55%) across all NHL subtypes and (90)Y doses, even in poor-risk categories (refractory to last anti-CD20-containing regimen, 73% [CR/CRu, 60%]; bulky disease: 71% [CR/CRu, 43%]). Patients with prior ASCT received lower doses, but achieved an OR rate of 41% (CR/CRu, 29%). For patients with follicular lymphoma (FL), OR rates and median PFS increased with total (90)Y-dose, reaching 100% (CR/CRu, 92%) and 24.6 months, respectively, at the highest dose levels (> 30 mCi/m(2) total (90)Y-dose [1,110 MBq/m(2)]). Further, patients with FL refractory to prior anti-CD20-containing regimens achieved 90% (nine of 10 patients) OR and CR/CRu rates and a median PFS of 21.5 months., Conclusion: Fractionated anti-CD22 radioimmunotherapy provides high total doses of (90)Y, yielding high rates of durable CR/CRus in relapsed/refractory NHL, resulting in 20 mCi/m(2) x 2 weeks as the recommended dose for future studies.

Protein tyrosine kinases of the Janus kinase (JAK) family are associated with many cytokine receptors, which, on ligand binding, regulate important cellular functions such as proliferation, survival, and differentiation. In multiple myeloma, JAKs may be persistently activated due to a constant stimulation by interleukin (IL)-6, which is produced in the bone marrow environment. INCB20 is a synthetic molecule that potently inhibits all members of the JAK family with a 100- to 1,000-fold selectivity for JAKs over >70 other kinases. Treatment of multiple myeloma cell lines and patient tumor cells with INCB20 resulted in a significant and dose-dependent inhibition of spontaneous as well as IL-6-induced cell growth. Importantly, multiple myeloma cell growth was inhibited in the presence of bone marrow stromal cells. The IL-6 dependent cell line INA-6 was particularly sensitive to the drug (IC50<1 micromol/L). Growth suppression of INA-6 correlated with an increase in the percentage of apoptotic cells and inhibition of signal transducer and activator of transcription 3 phosphorylation. INCB20 also abrogated the protective effect of IL-6 against dexamethasone by blocking phosphorylation of SHP-2 and AKT. In contrast, AKT phosphorylation induced by insulin-like growth factor-I remained unchanged, showing selectivity of the compound. In a s.c. severe combined immunodeficient mouse model with INA-6, INCB20 significantly delayed INA-6 tumor growth. Our studies show that disruption of JAKs and downstream signaling pathways may both inhibit multiple myeloma cell growth and survival and overcome cytokine-mediated drug resistance, thereby providing the preclinical rationale for the use of JAK inhibitors as a novel therapeutic approach in multiple myeloma.

Multiple myeloma (MM) is a rapidly fatal plasma-cell malignancy that evolves mainly in the bone marrow. Melphalan is widely used to treat patients with MM but as yet its mechanisms of action are poorly documented. In the current study, we demonstrate that melphalan induces a drastic downregulation of Mcl-1L, Bcl-x(L) and BimEL in human melphalan-sensitive myeloma cells while the most potent proapoptotic isoforms, BimL and S, are affected to a lesser extent. Moreover, Mcl-1L and BimEL disappearance is associated with the generation of proapoptotic cleaved forms generated by a caspase cleavage. In myeloma cells, we have previously shown that Mcl-1 neutralizes the proapoptotic function of Bim and therefore, prevents the activation of death effectors. In this study, we demonstrate that melphalan disrupts the Mcl-1/Bim complex whereas the Bcl-2/Bim complex is not modified. The disappearance of full length Mcl-1 allows the release of Bim isoforms, particularly L and S, which can exert their proapoptotic function and leads to Bax activation and cytochrome c release. Thus, we can hypothesize that the cleaved 26 kDa proapoptotic Mcl-1 and the 19 and 12 kDa of Bim, generated during melphalan treatment could contribute to the amplification loop of apoptosis.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) is a small-molecule, selective, noncompetitive inhibitor directed against human IMPDH. In this report, we show that VX-944 inhibits in vitro growth of human multiple myeloma (MM) cell lines via induction of apoptosis. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs) do not protect against VX-944-induced MM cell growth inhibition. VX-944 induced apoptosis in MM cell lines with only modest activation of caspases 3, 8, and 9. Furthermore, the pan-caspase inhibitor z-VAD-fmk did not inhibit VX-944-induced apoptosis and cell death. During VX-944-induced apoptosis, expressions of Bax and Bak were enhanced, and both apoptosis-inducing factor (AIF) and endonuclease G (Endo G) were released from the mitochondria to cytosol, suggesting that VX-944 triggers apoptosis in MM cells primarily via a caspase-independent, Bax/AIF/Endo G pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin and melphalan even in the presence of BMSCs. Taken together, our data demonstrate a primarily non-caspase-dependent apoptotic pathway triggered by VX-944, thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional agents which trigger caspase activation.

Previous studies have demonstrated the in vitro and in vivo activity of CC-5013 (Revlimid), an immunomodulatory analog (IMiD) of thalidomide, in multiple myeloma (MM). In the present study, we have examined the anti-MM activity of rapamycin (Rapamune), a specific mTOR inhibitor, combined with CC-5013. Based on the Chou-Talalay method, combination indices of less than 1 were obtained for all dose ranges of CC-5013 when combined with rapamycin, suggesting strong synergism. Importantly, this combination was able to overcome drug resistance when tested against MM cell lines resistant to conventional chemotherapy. Moreover, the combination, but not rapamycin alone, was able to overcome the growth advantage conferred on MM cells by interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), or adherence to bone marrow stromal cells (BMSCs). Combining rapamycin and CC-5013 induced apoptosis of MM cells. Differential signaling cascades, including the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3'-kinase/Akt kinase (PI3K/Akt) pathways, were targeted by these drugs individually and in combination, suggesting the molecular mechanism by which they interfere with MM growth and survival. These studies, therefore, provide the framework for clinical evaluation of mTOR inhibitors combined with IMiDs to improve patient outcome in MM.

R115777, a nonpeptidomimetic farnesyl transferase inhibitor has recently demonstrated a significant antileukemic activity in vivo in acute myeloid leukemia. Multiple myeloma (MM) is a fatal hematological malignancy characterized by an accumulation of long-lived plasma cells within the bone marrow. In the present study, we have investigated the effect of the R115777 on growth and survival of myeloma cells. We have found that R115777 induced (1) a significant and dose-dependent growth inhibition of the three myeloma cell lines tested; and (2) a significant and time-dependent apoptosis. R115777 also induced apoptosis in the bone marrow mononuclear cell population of four MM patients, being almost restricted to the malignant plasma cells. Finally, we have investigated the effect of the R115777 in the Ras/MAPK and JAK/STAT pathways which are implicated in survival and/or proliferation in MM. The phosphorylation of both STAT3 and ERK1/2 induced by IL-6 was totally blocked at 15 microM of R115777 and partially blocked when R115777 was used at 10 and 5 microM. The induction of apoptosis by R115777 in myeloma cells and its implication in the regulation of JAK/STAT signalling suggest that R115777 might be an interesting therapeutical approach in MM.

We retrospectively evaluated the role of rituximab (R) in maintenance treatment after autologous stem cell transplantation performed in patients with relapsed follicular lymphoma. We compared the outcome of 67 follicular lymphoma (FL) patients according to the use of rituximab maintenance (RM) or not. All patients received rituximab plus chemotherapy before autologous stem-cell transplantation (ASCT). Patients received median of two lines of prior therapy. The RM schedule was one injection of rituximab every 3 months for 2 years. Median follow-up is 4.6 years. The 3-year progression-free survival (PFS) after ASCT was 86 % with RM vs. 46 % without (p = 0.0045). Median is not reached in the RM arm vs. 31 months in non-RM arm. The 3-year OS was 96 % with RM vs. 78 % without (p = 0.059). The present monocentric study shows that 2 years of RM after ASCT significantly increases response duration for non-naive rituximab relapsed FL patients compared with observation. [ABSTRACT FROM AUTHOR]

Background Pixantrone is an aza-anthracenedione with enhanced, preclinical antitumor activity and reduced cardiotoxicity compared with doxorubicin. Patients and methods We compared the efficacy and toxic effect of CPOP-R (substituting pixantrone for doxorubicin) against CHOP-R in untreated, diffuse large B-cell lymphoma (DLBCL) patients. The primary objective was to demonstrate non-inferiority of CPOP-R by complete response/complete response unconfirmed (CR/CRu) rate. Results The CR/CRu rate for CPOP-R was 75% versus 84% for CHOP-R. Three-year overall survival was lower for CPOP-R (69% versus 85%) (P = 0.029). Median progression-free survival (PFS) was not reached for CPOP-R and was 40 months for CHOP-R [HR 95% confidence interval (CI) = 1.02 (0.60, 1.76), P = 0.934]. Fewer CPOP-R patients developed congestive heart failure (CHF) (0% versus 6%, P = 0.120), ≥20% declines in ejection fraction (2% versus 17%, P = 0.004), or elevations in troponin-T (P = 0.003). Conclusions CPOP-R is an active regimen with modestly lower response rates than CHOP-R but similar PFS and event-free survival. This study demonstrates a substantially lower cardiotoxicity of pixantrone compared with doxorubicin when used as first-line therapy in DLBCL. [ABSTRACT FROM PUBLISHER]