Multiple myeloma is a clonal plasma cell disorder in which growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type I (IGF-I). Bortezomib, the ...first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma.
We analyzed the impact of IGF-I combined with proteasome inhibitors on multiple myeloma cell lines in vivo and in vitro as well as on fresh human myeloma cells.
Our study shows that IGF-I enhances the cytotoxic effect of proteasome inhibitors against myeloma cells. The effect of bortezomib on the content of proapoptotic proteins such as Bax, Bad, Bak, and BimS and antiapoptotic proteins such as Bcl-2, Bcl-XL, XIAP, Bfl-1, and survivin was enhanced by IGF-I. The addition of IGF-I to bortezomib had a minor effect on NF-κB signaling in MM.1S cells while strongly enhancing reticulum stress. This resulted in an unfolded protein response (UPR), which was required for the potentiating effect of IGF-I on bortezomib cytotoxicity as shown by siRNA-mediated inhibition of GADD153 expression.
These results suggest that the high baseline level of protein synthesis in myeloma can be exploited therapeutically by combining proteasome inhibitors with IGF-I, which possesses a "priming" effect on myeloma cells for this family of compounds.
Multiple myeloma (MM) is a B cell neoplasm characterized by bone marrow infiltration with malignant plasma cells. IGF-1 signalling has been explored as a therapeutic target in this disease. We ...analyzed the effect of the IKK2 inhibitor AS602868, in combination with a monoclonal antibody targeting IGF-1 receptor (anti-IGF-1R) in human MM cell lines. We found that anti-IGF-1R potentiated the apoptotic effect of AS602868 in LP1 and RPMI8226 MM cell lines which express high levels of IGF-1R. Anti-IGF-1R enhanced the inhibitory effect of AS602868 on NF-κB pathway signalling and potentiated the disruption of mitochondrial membrane potential caused by AS602868. These results support the role of IGF-1 signalling in MM and suggest that inhibition of this pathway could sensitize MM cells to NF-κB inhibitors.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
AIM: To evaluate quantitatively and qualitatively the different CD34+cell subsets after priming by chemotherapy granulocyte colony-stimulating factor(± G-CSF)in patients with acute myeloid ...leukemia.METHODS: Peripheral blood and bone marrow sampleswere harvested in 8 acute myeloid leukemia patients during and after induction chemotherapy. The CD34/CD38 cell profile was analyzed by multi-parameter flow cytometry. Adhesion profile was made using CXC chemokine receptor 4(CXCR4)(CD184), VLA-4(CD49d/CD29) and CD47.RESULTS: Chemotherapy ± G-CSF mobilized immature cells(CD34+CD38 population), while the more mature cells(CD34+CD38lowand CD34+CD38+populations) decreased progressively after treatment. Circulating CD34+cells tended to be more sensitive to chemotherapy after priming with G-CSF. CD34+cell mobilization was correlated with a gradual increase in CXCR4 and CD47expression, suggesting a role in cell protection and the capacity of homing back to the marrow.CONCLUSION: Chemotherapy ± G-CSF mobilizes into the circulation CD34+bone marrow cells, of which, the immature CD34+CD38-cell population. Further manipulations of these interactions may be a means with which to control the trafficking of leukemia stem cells to improve patients’ outcomes.
AIM To evaluate the importance of the CD34+CD38-cell population when compared to the CD34+CD38+/low and CD34+CD38+/high leukemic cell sub-populations and to determine its correlations with leukemia ...characteristics and known prognostic factors, as well as with response to therapy and survival.METHODS Two hundred bone marrow samples were obtained at diagnosis from 200 consecutive patients with newly diagnosed acute myeloid leukemia(AML) were studied between September 2008 and December 2010 at our Institution(Hematology Department, Lyon, France). The CD34/CD38 cell profile was analyzed by multiparameter flowcytometry approach using 8 C panels and FACS CANTO and Diva software(BD Bioscience).RESULTS We analyzed CD34 and CD38 expression in bone marrow samples of 200 AML patients at diagnosis, and investigated the prognostic value of the most immature CD34+CD38-population. Using a cut-off value of 1% of CD34+CD38-from total “bulk leukemic cells” we found that a high(> 1%) level of CD34+CD38-blasts at diagnosis was correlated with advanced age, adverse cytogenetics as well as with a lower rate of complete response after induction and shorter disease-free survival. In a multivariate analysis considering age, leukocytosis, the % of CD34+ blasts cells and the standardized cytogenetic and molecular risk subgroups, a percentage of CD34+CD38-leukemic cells > 1% was an independent predictor of DFS HR = 2.8(1.02-7.73), P = 0.04 and OS HR = 2.65(1.09-6.43), P = 0.03.CONCLUSION Taken together, these results show that a CD34/CD38 “backbone” for leukemic cell analysis by multicolour flowcytometry at diagnosis provides useful prognostic information.
Abstract
Multiple Myeloma (MM) is a clonal plasma cell disorder whose growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type 1 (IGF-1). ...Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma and has been suggested to induce apoptotsis. We analyzed the impact of recombinant IGF-1 combined with the proteasome inhibitor bortezomib on human plasma cell lines in vitro and in vivo and on fresh human myeloma cells ex vivo. We found that IGF-1 enhanced the cytotoxic activity of bortezomib in vitro against the LP1, RPMI8226, U266 and MM1.S lines. This potentiating effect was confirmed on MM1.S cells using a flow cytometric analysis of annexin V staining, and showed that the enhanced toxicity could be inhibited by the presence of a monoclonal antibody directed against the IGF-1 receptor (IGF1-R). IGF-1 was also found to enhance the cytotoxic activity of other proteasome inhibitors against MM1.S cells, including MG115, MG132, PSI and epoximicin. In vivo studies were performed in SCID mice bearing MM1.S xenografts. The co-administration of IGF-1 bortezomib significantly delayed tumor growth in comparison to that observed in mice treated with bortezomib alone. Fresh human myeloma cells exposed to bortezomib ex vivo displayed a larger annexin V positive fraction when they were co-incubated with IGF-1 then when they were exposed to bortezomib alone. This effect, which could be observed in subpopulations of CD45 hi and CD45 lo cells, could be reversed by an antibody directed against IGF-1R. Thus in each of these situations, IGF-1 increased the sensitivity of multiple myeloma cells to the cytotoxic effect of bortezomib. Analysis of pro- and anti-apoptotic proteins in MM1.S cells by immunoblotting showed that the addition of IGF-1 to bortezomib significantly enhanced the content of Bax, Bad and Bak and significantly reduced the content of Bcl2, BclX-L and Bfl-1. Other observations made with the IGF-1/bortezomib combination include an increase in the content of cleaved caspase 3 and in P21 protein. Preliminary data showed an increased content of CHOP protein, suggesting that the IGF-1/bortezomib combination might enhance reticulum stress in MM1.S cells, thus leading to an Unfolded Protein Response (UPR) and to cell death. These results suggest that IGF-1 sensitizes myeloma cells to proteasome inhibitors by contributing to the enhancement of the reticulum stress. Overall these results suggest that exposure of myeloma cells to one of their key growth factors, IGF-1, significantly enhanced their sensitivity to bortezomib as well as to other proteasome inhibitors. This phenomenon appears to involve several pathways and may be dependent on the high baseline level of reticulum stress present in myeloma cells.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-2. doi:10.1158/1538-7445.AM2011-LB-2
Multiple Myeloma (MM) is a B cell neoplasm characterized by the accumulation of mature plasma cells (PCs) within the bone marrow (BM). Despite treatment advances, MM remains incurable in the vast ...majority of patients. Disease relapse typically occurs regardless of treatment with proteasome inhibitors (such as bortezomib and carfilzomib), immunomodulatory drugs (such as lenalidomide and pomalidomide), anti-CD38 monoclonal antibodies (such as daratumumab and isatuximab) or myeloablative melphalan and autologous stem cell transplantation, and also occurs following treatment with immunotherapeutics such as bispecific antibodies and/or CAR-T cells (targeting BCMA, FcRH5 or GPRC5D). This failure to achieve reliable cure in MM, despite the routine attainment of deep treatment responses against PCs, indicates the existence of important intra-tumor heterogeneity and the presence of rare drug-resistant MM cells with full tumorigenic capacity. To identify intra-clonal MM cell subpopulations, we examined BM samples from MM patients using a combination of FACS, single cell resolved immunoflourescence-FISH (IF-FISH), whole exome sequencing (WES), custom-capture targeted deep sequencing (CC-Seq) and single cell RNA sequencing (scRNAseq). We sought to characterize the genomic landscape and to track the dynamic interconnectedness of these intra-clonal subpopulations in patients over time. Using sequential FACS-IF-FISH studies of MM BM samples (n=140) we identified MM clone cellular subpopulations within the BM of MM patients that recapitulate the normal maturation stages between non-malignant post germinal centre B cells and mature PCs. These subpopulations include rare MM cells that resemble CD20 +CD38 -CD138 -Bcma -Irf4 - Xbp1s - B cells, CD20 -CD38 -CD138 - Bcma -Irf4 -Xbp1s - pre-plasmablasts and CD38 +CD138 -Bcma -/lowIrf4 -/+Xbp1s + pre-plasma cells, as well as predominating tumor-bulk CD38 +CD138 +Irf4 +Bcma +Xbp1s + plasma cells. Importantly, MM progenitor cells were typically found to possess all of the chromosomal translocations and copy number variations (CNV) present in MM PCs including whole chromosome ploidy changes, translocations such as t(11;14), t(4;14) and t(14;16), and secondary aberrations such as gain(1q), del(1p) and del(17p). To examine if MM progenitor cells also possess the single nucleotide variations (SNV) present within MM PCs, and are thus fully malignant, and to track MM progenitor subpopulations and their inter-relatedness within patients over time, we performed WES (n=60) plus targeted deep CC-Seq (n=210) on purified cell subpopulations from 17 patients, including 5 patients whom we sampled serially over >3 years. A median of 5 cellular subpopulations were isolated from each BM sample, enriched by factors of up to 1000-fold or more, and each subpopulation was then sequenced to a depth of 4,000-20,000x. Significantly, SNVs that were present in tumor-bulk MM PCs were also regularly detected in MM progenitor cells, although the low frequency of the clonal progenitor cells often prevented capture of complete SNV profiles. At the same time, analyses of serial BM samples from repeat donor patients taken pre- and post-treatment demonstrated that relapsing MM PCs typically did not appear to derive linearly from pre-treatment PCs, as the relapsing PCs commonly lacked a number of SNVs that had been present in pre-treatment PCs. Importantly, we instead observed that relapsed MM plasma cells appeared to consistently derive from MM progenitor cell populations, frequently B cells, as emergent relapse-specific SNVs that were exclusively detected in relapsing post-treatment PCs (but in not pre-treatment PCs) could often also be detected within pre-treatment CD138- CD38- progenitor cells isolated from BM samples that had been collected up to 3 years prior to relapse. We conclude that MM B cells and pre-plasma cell progenitors possess all of the clonal genomic aberrations required for full malignant potential. Tracking of these cells in vivo in patients over time by their SNV profile implicates them as the cellular origin of PC relapse and recurrent MM disease. We propose that cure of MM requires eradication of MM progenitor cells alongside plasma cells. We are currently conducting scRNA-seq and CITE-seq studies of primary MM samples to further characterize MM progenitor cells for gene and protein expression in order to define their optimal therapeutic targets.
Gain of chromosome 1q is a recurrent genomic feature of many human tumors and is present in 33-48% of newly diagnosed MMs (NDMM). Analogous to deletion of 17p, + 1q can occur as a secondary genetic ...event in all MM subtypes, and is associated with poor prognosis. Although uncommon in precursor stages such as monoclonal gammopathy of undetermined significance (MGUS), the prevalence of +1q rises significantly in symptomatic MM, and at relapse, suggesting roles in malignant transformation and treatment escape. The molecular mechanisms underlying these associations remain poorly understood. Here, we report an analysis of the transcriptional consequences of +1q in single MM cells and in bulk tumor samples.
To define the molecular consequences of + 1q at a cellular level, we performed single cell RNA-sequencing (scRNA-seq) and single-cell inferred CNV (sciCNV) analysis of primary MM bone marrow samples. This provided paired DNA/RNA omics information within single cells. From MM samples containing intra-clonal populations with and without +1q we directly examined the effects of +1q on gene expression. The effects of +1q were delineated using intra-clonal isogenic sibling cells lacking +1q as controls. We validated our findings using 10 tumor cohorts, including 2 MM cohorts representing >1300 NDMM patients and 8 non-MM cohorts representing 3,915 patients with other malignancies.
From these studies we show that primary MM cells with +1q significantly upregulate mitochondrial oxidative phosphorylation (OXPHOS), causing increased reactive oxygen species and reduced energy stress, compared with isogenic sibling cells without +1q. Despite increased OXPHOS, +1q cells do not appear to experience increased hypoxia nor reduced glycolysis, suggesting that they continue to benefit from aerobic glycolysis (Warburg metabolism). At the same time +1q MM cells overexpress ADAR and suppress IFN type 1 and 2 responses, repressing tumor immunity. Overexpression of CD46, CFH, CFHR1, CHFR5, ARPC5 and SELL from +1q further suppress pathways involved in tumor immune recognition, particularly by complement and antibodies. MM tumors overexpressing MYC are enriched amongst +1q samples, with MYC co-operating with MCL1 to promote mitochondrial oxidative phosphorylation, leading to apparent enrichment of a MYC gene expression signature in +1q samples, though MYC itself is not increased by +1q. From MM registry data, +1q cooperates with MYC to promote OXPHOS, and OXPHOS strongly predicts patient survival, particularly in high-risk t(4;14) and del(17p) subtypes. Examination of 8 non-MM tumor cohorts reveals that other human cancers with +1q similarly upregulate OXPHOS and MYC programs and/or suppress tumor immunity (IFN-g and -a responses, complement pathways and allograft rejection).
Overall, from these multi-omic analyses we identify critical reprogramming events in MM and other human cancers that arise from +1q and that predict patient survival.
Background: The addition of immunomodulatory (IMiDs) drugs to monoclonal antibodies targeting the transmembrane glycoprotein CD38 has demonstrated very encouraging and durable responses in myeloma ...patients. It is believed that the synergistic effects observed with anti-CD38 antibodies and IMiDs are derived from their co-modulation of the host adaptive and innate immunity and therefore it is plausible to speculate that acquired resistance to daratumumab and IMiDs may be largely immune-mediated. The aim of the present study was to 1) interrogate at the single cell level the bone marrow immune repertoire of daratumumab sensitive and resistant patients, 2) identify cellular mediators of resistance to anti-CD38 antibodies and 3) define potential means to reinstate sensitivity to daratumumab and IMiDs.
Methods and Results: Serial BM aspirates (n=44) were collected from patients treated with single agent daratumumab (MMY3012 trial) or daratumumab + pomalidomide (MM014 trial) prior to initiation of therapy, C3D1 and at relapse. Bone marrow mononuclear fractions were isolated through ficoll density gradients coupled with magnetic sorting of CD138pos and CD138neg cells. Unbiased mRNA profiling of BM CD138neg cells was performed by single-cell RNA-seq (scRNA-seq) using the GemCode system (10x Genomics). Paired-end sequencing was performed on Illumina NEXTseq and NOVAseq platforms. Cell Ranger Single and Seurat were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting and data analysis. Sequencing data were analyzed by principal component analysis (PCA), clustering with multi-sample batch correction and then visualized by t-distributed stochastic neighbor embedding (t-SNE) projection. Comparison of the single cell transcriptomes of CD138neg cells from responding patients pre- and post- treatment revealed that Daratumumab and Pomalidomide dramatically modify the immune cells composition (immunome) of the bone marrow niches leading to: 1) expansion of effector T cells (KLRG1high, GZMAhigh, CCL5high), 2) significant depletion of CD38high / FCGR3Ahigh NK cells with retained population of cytotoxic NK cells (CD27high, KLRB1high, NCR3high, GZMApos, PRF1pos), 3) depletion of FCGR3Ahigh / CD14low monocytes, 4) expansion of M1 inflammatory macrophages and depletion of plasmacytoid dendritic cells. Similar changes were seen in patients treated with single agent daratumumab (without IMiDs) however with a lesser expansion of effector T cells and in particular reduced marrow infiltrating inflammatory macrophages. In contrast, the immunome of daratumumab and pomalidomide resistant patients was characterized by a reduced central memory T cells (TCM), and a largely exhausted effector T cells populations that are CD28neg and expressing checkpoint inhibitors (LAG3and TIGIT significantly more than PDCD1) as well as high expression of TIM3 (HAVCR2) on marrow macrophages. Upregulation of LAG3 and TIGIT expression on T cells was also confirmed at the antigenic level by flow cytometry. Consistent with the non-bystander and suppressive effect of LAG3 and TIGIT on the function of effector T cells, activation (CD107a expression) and proliferation of LAGpos and/or TIGITpos sorted bone marrow T cells from resistant patients were significantly reduced in response to autologous myeloma cells stimulation or CD3/CD28 crosslinking. Lastly, a higher proportion and number of clonal T cell (through single cell TCR sequencing) was also observed in responding (≥ PR) vs non-responding (< PR) patients. Interrogation of the myeloma cells transcriptome, showed little to no loss of CD38 transcript at the time of acquired resistance with rather upregulation of complement inhibitory molecule CD59 and NFκB signature genes.
Conclusion: A systematic unsupervised interrogation of the bone marrow immunome of daratumumab and IMiDs treated MM patients demonstrated a significant activation of adaptive and innate immunity in responding patients and revealed an expansion of exhausted T cells with upregulation of the checkpoint inhibitors LAG3 and TIGIT in resistant patients. Our findings warrant the exploration of LAG3- and/or TIGIT-blocking strategies as potential means to reinstate sensitivity to daratumumab and IMiDs in myeloma patients.
Neri:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. McCulloch:Celgene: Honoraria; Takeda: Other: Travel expenses. Thakurta:Celgene Corporation: Employment, Equity Ownership. Bahlis:Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding.
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