Non-myeloma Cells Research Articles

Towards the Segmentation and Classification of White Blood Cell Cancer Using Hybrid Mask-Recurrent Neural Network and Transfer Learning.

Inside the bone marrow, plasma cells are created, and they are a type of white blood cells. They are made from B lymphocytes. Antigens are produced by plasma cells to combat bacteria and viruses and prevent inflammation and illness. Multiple myeloma is a plasma cell cancer that starts in the bone marrow and causes the formation of abnormal plasma cells. Multiple myeloma is firmly identified by examining bone marrow samples under a microscope for myeloma cells. To diagnose myeloma cells, pathologists have to be very selective. Furthermore, because the ultimate decision is based on human sight and opinion, there is a possibility of error in the result. The nobility of this research is that it provides a computer-assisted technique for recognizing and detecting myeloma cells in bone marrow smears. For recognizing purposes, we have used Mask-Recurrent Convolutional Neural Network, and for detection purposes, Efficient Net B3 has been used. There are already many studies on white blood cell cancer, but very few with both segmentation and classification. We have designed two models. One is for recognizing myeloma cells, and the other is for differentiating them from nonmyeloma cells. Also, a new data set has been made from the multiple myeloma data sets, which has been used in our classification model. This research focuses on hybrid segmentation models and increases the accuracy level of the classification model. Both of our models are trained pretty well, where the Mask-RCNN model gives a mean average precision (mAP) of 93% and the Efficient Net B3 model gives 94.68% accuracy. The result of this research indicates that the Mask-RCNN model can recognize multiple myeloma and Efficient Net B3 can distinguish between myeloma and nonmyeloma cells and beats most of the state of the art in myeloma recognition and detection.

Combined Inhibition of Chk1 and Wee1 As a New Therapeutic Strategy for High-Risk Multiple Myeloma Patients

Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite introduction of novels agents that have significantly improved clinical outcomes, most patients relapse and develop drug resistance. Therefore, novel therapeutic strategies to overcome chemotherapy resistance are needed in clinical settings.MM is characterized by genomic instability and high level of replicative stress that arise during the disease's pathogenesis. In response to replicative and DNA damage stress, MM cells activate various DNA damage signaling pathways that include the kinases ATR, Chk1 and Wee1. Chk1 is activated by ATR after DNA damage or replicative stress and promote cell cycle arrest through the regulation of S and G2 checkpoints. Wee1 kinase plays a central role in the proper timing of cell division cycle controlling entry into mitosis and DNA replication during S phase. Interestingly, experimental studies have identified that Chk1 inhibitors are strongly synergistic with Wee1 inhibitors in various cancer models. Furthermore, Chk1 and Wee1 inhibitors are currently under clinical investigation.Here, we investigated the therapeutic interest of Chk1 and Wee1 in MM.First we investigated the prognostic value of Chk1 and/or Wee1 gene expression in MM patients. Both Chk1 and Wee1 were significantly overexpressed in human myeloma cell lines (HMCLs, n= 40) compared to bone marrow plasma cells (BMPCs, n=5) (P<001 and P<001 respectively). Chk1 and Wee1 expression were significantly higher in the poor prognosis “proliferation” MM subgroup (P<001 and P<001 respectively).High Chk1 expression and high Wee1 expression could predict for shorter overall survival (OS) in 4 independent cohorts of newly-diagnosed patients (P<0.001 and P=0.007 in the HM cohort (N=206), P<0.001 and P=0.008 in UAMS-TT2 cohort (N=345), P<0.001 and P<0.001 in Hovon cohort (N=282) and P=0.002 and P<0.001 in UAMS-TT3 cohort (N=186)). Same results were obtained for event free survival (EFS). Moreover, Chk1 and Wee1 high expression are associated with a poor prognosis in a cohort of 242 patients treated by Bortezomib monotherapy (Mulligan Cohort, P<0.001 and P=0.001 respectively).Interestingly, combined high expression of Chk1 and Wee1 are associated to significantly shorter survival compared to high expression of Chk1 or Wee1 alone in the 4 independent cohorts investigated. Same results were obtained for event free survival (EFS).According, to these results, we investigated the therapeutic interest of Chk1 and Wee1 inhibitors (AZD7762 and MK-1775) alone and in combination in MM.Chk1 inhibitor (AZD7762) induced a dose dependent cell growth inhibition in 13 HMCLs with a median IC50 of 179nM (range 69-366nM). A correlation between HMCLs sensitivity and P53 mutation was observed (r =0.7, P<0.01).Wee1 inhibitor (MK-1775) induced also a dose dependent inhibition of cell growth in all investigated HMCLs (n=10) with a median of IC50 of 450nM (range 100-800nM).Combination treatment of Wee1 inhibitor IC20 with various doses of Chk1 inhibitor was performed in 4 HMCLs. We identified a high synergy (combination index range 0.4-0.7) associated with a significant and remarkable reduction of Chk1 inhibitor (AZD7762) IC50 (198nM to 20nM for XG6; 166nM to 25nM for AMO1; 386nM to 18nM for XG7 and 163nM to 21nM for OPM2) in all the tested HMCLs. Chk1 and Wee1 inhibitors combination demonstrated a significant increase in apoptosis induction, proliferation inhibition and DNA damage induction compared to Chk1i or Wee1i alone in 3 HMCLs.We investigated the effects of Chk1i, Wee1i and Chk1i/Wee1i combination on primary MM cells of patients (n = 5) co-cultured with their bone marrow microenvironment. After 4 days of treatment, cells were enumerated and the fraction of viable myeloma cells (CD138+) and non-myeloma cells (CD138-) were determined by flow cytometry. Chk1i/Wee1i combination significantly and specifically induced a higher toxicity on myeloma cells of patients compared to Chk1i or Wee1i alone. This toxicity is specific of MM cells since normal bone marrow cells were significantly less affected by the combination treatment (P < 0.01).Taken together, these data suggest that association of Chk1i and Wee1i may represent a promising therapeutic approach in high-risk MM patients characterized by high Chk1 and Wee1 expression. DisclosuresBruyer:Diag2tec: Employment. Martin:Diag2tec: Employment. Cartron:Celgene: Consultancy, Employment; Sanofi, BMS, Jansen, celgene, Roche, Gilead: Equity Ownership; Roche: Consultancy, Equity Ownership, Honoraria, Research Funding. Hose:EngMab: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding.

Generation of a Lenalidomide-Sensitive Mouse Model for Multiple Myeloma By Expression of the Crbn I391V Mutant in Mopc-315.BM Cells

Lenalidomide is a highly active treatment for multiple myeloma. We and others have previously shown that lenalidomide targets Ikaros (IKZF1) and Aiolos (IKZF3) for ubiquitination and degradation via the cereblon (CRBN) E3 ubiquitin ligase. Depletion of these lymphoid transcription factors selectively inhibits growth of multiple myeloma cells. In contrast to human cells, mouse cells are insensitive to lenalidomide due to a single amino acid change in the murine CRBN (mCRBN) protein (isoleucine instead of valin at position 391). Therefore, lenalidomide could so far not be studied in syngenic mouse multiple myeloma models. The mouse MOPC-315.BM cell line engrafts in the bone marrow of BALB/c mice and induces multiple myeloma after intravenous injection (Riedel et al., PLoS one 2012 and Hofgaard et al., PLoS one 2012). We aimed to generate a lenalidomide-sensitive mouse model by retroviral expression of a mutant murine Crbn where the isoleucine at position 391 is replaced by valin (mCrbnI391V) in MOPC-315.BM cells.Western blot analyses showed that MOPC-315.BM multiple myeloma cells express IKZF1 and IKZF3 proteins at a high level as compared to non-myeloma cell lines. Expression of a dominant negative IKZF3 isoform that inactivates both, IKZF3 and IKZF1, inhibited proliferation of MOPC315.BM cells, demonstrating that these cells depend on IKZF3 and IKZF1. In unmodified or mCrbn -overexpressing MOPC-315.BM cells even high concentrations (>10 µM) of lenalidomide did not induce degradation of IKZF1 and IKZF3 and consistently had no effect on proliferation. In contrast, we observed effective degradation of IKFZ1 and IKZF3 in MOPC-315.BM expressing mCrbnI391V and these cells were highly sensitive to lenalidomide, pomalidomide, and thalidomide. Having shown that expression of mCrbnI391V sensitizes mouse multiple myeloma cells in vitro, we sought to investigate whether this is also true in vivo .For that purpose, we injected mCrbnI391V-expressing MOPC-315.BM cells in BALB/c mice. Luciferase expression of MOPC-315.BM cells allowed us to monitor tumor growth non-invasively by bioluminescense at different time points. Similar to the unmodified MOPC-315.BM cells, we detected mCrbnI391V-expressing MOPC-315.BM in the bone between days 12 to 19 by bioluminescense and continuous growth of the lesions until mice became sick around day 40. This demonstrated that expression of mCrbnI391V did not affect the potential of MOPC-315.BM to induce multiple myeloma in vivo. We next treated the mice with different dosages and schedules of lenalidomide by intraperitoneal injection when tumor engraftment was clearly measurable by bioluminescense in all mice. Lenalidomide at a high concentration of 25 mg/kg given twice weekly had only weak effects on tumor growth. In contrast, daily treatment with low concentrations (1 mg/KG) effectively inhibited tumor growth, demonstrating that continuous inactivation of IKZF1 and IKZF3 is necessary for lenalidomide activity in vivo .In conclusion, our results demonstrate that mouse multiple myeloma cells can be sensitized to lenalidomide by expression of mCrbnI391V in vitro and in vivo . This model will allow for the first time to test drug combinations including lenalidomide in a syngenic mouse model. Furthermore, it serves as a proof-of-concept for the generation of transgenic lenalidomide-sensitive mouse models for multiple myeloma and other hematologic malignancies in the future. [Display omitted] DisclosuresKronke:Celgene: Honoraria, Other: Travel Support; Takeda: Consultancy, Other: Travel Support.

Bovine pestivirus is a new alternative virus for multiple myeloma oncolytic virotherapy

BackgroundThe oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients’ antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46.MethodsWe treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines.ResultsHuman myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle.ConclusionsOverall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.

Graft-Versus-Host Disease in Multiple Myeloma Patients Treated With Daratumumab After Allogeneic Transplantation

IntroductionAllogeneic hematopoietic cell transplantation (allo-HCT) represents an adoptive immunotherapy strategy for eliciting a graft-versus-myeloma, the effect for high-risk or relapsed multiple myeloma (MM). Allo-HCT recipients are at risk for graft-versus-host disease (GVHD) as well as associated increases in morbidity and mortality. Daratumumab, an anti-CD38 human immunoglobulin G1 kappa humanized monoclonal antibody, is used for treatment of MM. Daratumumab also affects CD38+ nonmyeloma cells, including T cells, which mediate GVHD. The use of daratumumab after allo-HCT has not been well described, and its potential impact on GVHD is unknown. Patients and MethodsIn a multicenter retrospective study, we evaluated incidence of GVHD in 34 patients with relapsed MM treated with daratumumab after allo-HCT. ResultsOverall response to daratumumab (partial response or better) was 41% (95% confidence interval, 24-59). Five patients (15%) developed acute GVHD after daratumumab therapy; no chronic GVHD events were observed after daratumumab therapy. One of these 5 patients had a history of chronic GVHD and developed a flare of acute GVHD during daratumumab therapy. The remaining 4 patients did not have a history of GVHD before daratumumab. ConclusionThe incidence of GVHD after daratumumab was low and did not result in increased exacerbation of GVHD in patients with a history of GVHD.

Expression of CRBN Signaling Pathway Proteins Assessed By Immunohistochemical Staining As Candidate Biomarkers for Outcome in Myeloma Patients Treated with IMiDs

Cereblon (CRBN), Ikaros (IKZF1), Aialos (IKZF3) and multiple myeloma oncogene 1 (MUM1) are important component of CRBN signaling pathway when treat MM cells with IMiDs. CRBN interacts with the DNA damage-binding protein-1 (DDB1), Cullin 4 (Cul4A or Cul4B) and regulator of Cullins 1 (ROC1) to form the functional E3 ubiquitin ligase complex (E3ULC). CRBN increases the interaction between E3ULC and IKZF1/3, leading to increased ubiquitination degradation of IKZF1/3, and then induce cytotoxicity in myeloma cells. Subsequently, degradation of IKZF1/3 induce depression of multiple myeloma oncogene 1 (MUM1), which is also called interferon regulatory factors (IRF4) and proved to be involved in the anti-MM activity of IMIDs in previous studies. Immunohistochemical (IHC) staining may be a convenient approach for researchers to differentiate the myeloma cells and non-myeloma cells in BM samples. In this study, we evaluated CRBN, IKZF1/3 and MUM1 expression level in bone marrow (BM) by immunohistochemical (IHC) staining and investigated the relationship between expression level and treatment outcome after IMiDs-based or bortezomib-based therapy in 123 newly diagnosed multiple myeloma (NDMM) patients. H-score method was applied according to both intensity and extent of staining. The intensity was graded from 0 to 3(“0”for absent staining, “1” for weak expression, “2” for intermediate expression, and “3” for strong expression of the protein). The extent was graded from “0” to “100” to represent the percentage of MM cells with positive staining of any intensity. H-score was obtained by multiplying the intensity and extent score, ranging from 0 to 300, which reflected protein expression level in MM cells. The median H-score of CRBN, IKZF1, IKZF3 and MUM1 were 200, 0, 180 and 180, respectively. According to the median H-score, we classified the patients into high or low expression group. One hundred and twenty-three NDMM patients were enrolled in this study, including 64 males (52.0%) and 59 females (48.0%). The median age was 60 years (range 34-84). Fifty-one patients (41.5%) received IMiDs-containing regimen as the first-line therapy. The median follow-up time was 24.0 months (range, 10-76 months). After treated with IMiDs, patients with high level of CRBN and MUM1 achieved better overall response rate (ORR) than those expressed low level (CRBN, 88.0% vs. 42.3%, P=0.001; MUM1, 83.3% vs. 48.1%, P=0.009). Besides, patients with CRBN and MUM1 overexpression also had better overall survival (median OS, CRBN, not reached vs. 21.0 months, P=0.004; MUM1, not reached vs. not reached, P=0.021) and progression free survival (median PFS, CRBN, 28.0 vs. 12.0 months, P=0.002; MUM1, 32.0 vs. 12.0 months, P<0.001) than patients with low level, as well as 2-year OS rate (CRBN, 86% vs. 44%, P=0.005; MUM1, 81% vs. 51%, P=0.003) and PFS rate (CRBN, 66% vs. 17%, P=0.001; MUM1, 63% vs. 20%, P<0.001). In addition, in high CRBN and MUM1 expression group, patients treated with IMiDs had a higher 2-year PFS rate than Bortezomib presentation (CRBN, 66% vs. 45%, P=0.004; MUM1, 63% vs. 36%, P=0.003). In low CRBN and MUM1 expression group, patients treated with IMiDs had an inferior OS (median OS, CRBN, 21.0 vs. 57.0 months, P=0.001; MUM1, not reached vs. 57.0 months, P<0.001) and PFS (median PFS, CRBN, 12.0 vs. 31.0 months, P=0.003; MUM1, 12.0 vs. 32.0 months, P<0.001) than patients with Bortezomib, as well as 2-year OS rate (CRBN, 44% vs. 91%, P=0.002; MUM1, 51% vs. 100%, P<0.001) and PFS rate (CRBN, 17% vs. 59%, P=0.010; MUM1, 20% vs. 68%, P<0.001). This study identified high levels of CRBN pathway proteins CRBN and MUM1 were correlated with favorable ORR and survival in NDMM patients with IMiDs therapy, also suggested that expression levels of CRBN signaling pathway proteins in plasma cells assessed by IHC staining could better direct clinic individualized therapy. DisclosuresNo relevant conflicts of interest to declare.

Graft-versus-host disease (GVHD) risk with daratumumab (Dara) therapy post allogeneic transplantation (alloHCT) for multiple myeloma (MM).

8028Background: Daratumumab (Dara) is an antibody targeting CD38+ MM cells that also affects CD38+ non-myeloma cells, including T cells, which mediate GVHD. Regulatory T cells (Tregs) are reduced w...

SET8 Is a Potential Therapeutic Target in MM

Epigenetic regulation mechanisms - such as histone marks, DNA methylation and miRNA - are often misregulated in cancers and are associated with tumorigenesis and drug resistance. Multiple Myeloma (MM) is a malignant plasma cell disease that accumulates within the bone marrow. Epigenetic modifications in MM are associated not only with cancer development and progression, but also with resistance to chemotherapy. This epigenetic plasticity can be targeted with epidrugs, nowadays used in treatment of several cancers.We recently identified a significant overexpression of the lysine histone methyltransferase SETD8 in MM cells (HMCLs; N=40) compared with normal plasma cells (N=5) (P<0.001). SETD8 (also known as SET8, PR-Set7, KMT5A) is the sole enzyme responsible for the monomethylation of histone H4 at lysine 20 (H4K20me1) which has been linked to chromatin compaction and cell-cycle regulation. In addition, SETD8 induces the methylation of non-histone proteins, such as the replication factor PCNA, the tumor suppressor P53 and its stabilizing protein Numb. While SETD8-mediated methylation of P53 and Numb inhibits apoptosis, PCNA methylation upon SETD8 enhances the interaction with the Flap endonuclease FEN1 and promotes cancer cell proliferation. SETD8 is also implicated in DNA damage response, helping 53BP1 recruitment at DNA double-strand breaks. Consistent with this, overexpression of SETD8 is found in various types of cancer and has been directly implicated in breast cancer invasiveness and metastasis. A role of SETD8 in development of MM has however never been described.We found that high SETD8 expression is associated with a poor prognosis in 2 independent cohorts of newly diagnosed patients (UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N=158). Specific SETD8 inhibition with UNC-0379 inhibitor, causing its degradation and H4K20me1 depletion, leads to significant growth inhibition of HMCLs (N=10) and the murine cell lines 5T33MM and 5TGM1. MM cells treated with UNC-0379 presented a G0/G1 cell cycle arrest after 24h of treatment, followed by apoptosis 48h later. To confirm that SETD8 inhibition is as efficient on primary MM cells from patients, primary MM cells (N=8) were co-cultured with their bone marrow microenvironment and recombinant IL-6 and treated for 4 days with UNC-0379. Interestingly, treatment of MM patient samples with UNC-0379 reduces the percentage of myeloma cells (65%; P<0.005) without significantly affecting the non-myeloma cells, suggesting a specific addiction of primary myeloma cells to SETD8 activity. Melphalan is an alkylating agent commonly used in MM treatment. As SETD8 is known to be involved in the DNA damage response, we investigated the effect of its combination with Melphalan on HMCLs. Results show that this particular drug combination strongly enhances double strand breaks in HMCLs monitored using 53BP1 foci formation and gH2AX detection. This result emphasizes a potential role of SETD8 in DNA repair in MM cells. Furthermore, GSEA analysis of patients with high SETD8 expression highlighted a significant enrichment of genes involved in DNA repair, MYC-MAX targets and MAPK pathway.Our study is the first to demonstrate the importance of SETD8 for MM cells survival and suggest that SETD8 inhibition represent a promising strategy to improve conventional treatment of MM with DNA damaging agents. DisclosuresNo relevant conflicts of interest to declare.

Combination Therapy with Bortezomib or Carfilzomib and Selinexor Induces Nuclear Localization of Ikbα and Overcomes Acquired Proteasome Inhibitor Resistance in Human Multiple Myeloma

Introduction: Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired-resistance in MM.Materials and Methods: PI-resistant myeloma cell lines, RPMI8226-B25 and U226 PSR, and their respective parental cell lines RPMI8226 and U266, were treated both in vitro with selinexor/bortezomib or selinexor/carfilzomib and assayed for apoptosis. In vivo studies using U266 and U266PSR tumors were performed in NOD/SCID-gamma (NSG) mice. Mice were treated with selinexor/bortezomib and single agents. Bone marrow biopsies from refractory myeloma patients were treated ex vivo with selinexor/bortezomib or selinexor/carfilzomib and assayed for apoptosis. Mechanistic studies included NFkB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry and proximity-ligation assay. IkBα knockdown and NFkB transcriptional activity were measured in selinexor/bortezomib treated MM cells.Results: We found that selinexor restored sensitivity of PI-resistant RPMI8226-B25 and U266PSR MM cells to bortezomib (P = 0.00055) and carfilzomib (P = 0.0017). Bortezomib, when combined with selinexor reduced U266 MM tumor growth versus single-agent bortezomib (P = 0.022) in NSG mice. NSG mice challenged with PI-resistant U266PSR MM tumors also had reduced tumor growth with selinexor/bortezomib as compared to single agent bortezomib (P = 0.0006). Combining bortezomib and selinexor improved survival in mice with U266 MM tumors (P = 0.0072) and PI-resistant U266PSR when compared to single-agent bortezomib (P = 0.0072). Myeloma cells from PI-refractory MM patients (n=14) were sensitized by selinexor to bortezomib (P = 0.002) and carfilzomib (P = 0.001) without affecting non-myeloma cells. Immunofluorescence microscopy of PI-resistant human MM cell lines found a greater than 212% increase in IkBα when compared to untreated cells (confirmed by Western blot). A similar increase in IkBα immunofluorescence was found in newly diagnosed, relapsed and refractory patient MM cells. ImageStream analyses of MM cells showed an increase in total and nuclear IkBα from selinexor/bortezomib exposure. Proximity-ligation assays showed that IkBα-NFkB-complexes were increased 12-fold in bortezomib/selinexor treated MM cells. IkBα knockdown abrogated selinexor/bortezomib induced cytotoxicity in MM cells. Selinexor/bortezomib treatment decreased NFkB transcriptional activity in addition to a reduction of NFkB induced IAP-1, IAP-2, BCL-2, cyclin D2 and c-myc protein expression..Conclusions: Selinexor, when used with bortezomib or carfilzomib has the potential to overcome proteasome-inhibitor drug-resistance in MM. Sensitization may be due to inactivation of the NFkB pathway by IkBα. Selinexor, an orally active selective inhibitor of XPO1-mediated nuclear export (SINE), is currently undergoing phase I/II studies in a variety of indications, including a combination with carfilzomib, in both relapsed and refractory MM patients (NCT02199665). The results presented in this study support combinatorial clinical trials in relapsed and refractory MM that utilize PI therapies. DisclosuresKashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Shain:Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Signal Genetics: Research Funding; Novartis: Speakers Bureau; Amgen/Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder.

XPO1 inhibitor combination therapy with bortezomib or carfilzomib induces nuclear localization of IκBα and overcomes acquired proteasome inhibitor resistance in human multiple myeloma.

Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired resistance in MM. PI-resistant myeloma cell lines both in vitro and in vivo and refractory myeloma patient biopsies were treated with selinexor/bortezomib or carfilzomib and assayed for apoptosis. Mechanistic studies included NFκB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assays. IκBα knockdown and NFκB activity were measured in selinexor/bortezomib-treated MM cells. We found that selinexor restored sensitivity of PI-resistant MM to bortezomib and carfilzomib. Selinexor/bortezomib treatment inhibited PI-resistant MM tumor growth and increased survival in mice. Myeloma cells from PI-refractory MM patients were sensitized by selinexor to bortezomib and carfilzomib without affecting non-myeloma cells. Immunofluorescence microscopy, Western blot, and ImageStream analyses of MM cells showed increases in total and nuclear IκBα by selinexor/bortezomib. Proximity ligation found increased IκBα-NFκB complexes in treated MM cells. IκBα knockdown abrogated selinexor/bortezomib-induced cytotoxicity in MM cells. Selinexor/bortezomib treatment decreased NFκB transcriptional activity. Selinexor, when used with bortezomib or carfilzomib, has the potential to overcome PI drug resistance in MM. Sensitization may be due to inactivation of the NFκB pathway by IκBα.

Identification of FAM46C As a Multiple Myeloma Repressor

Introduction: Partial loss of chromosome arm 1p frequently occurs in multiple myeloma (MM), and is associated with a poor prognosis. Several minimally altered regions on 1p have been identified, including 1p32.3, 1p31.3, 1p22.1-1p21.3, and 1p12. Cytoband 1p12 was deleted in 19% of cases, and this deletion was associated with shorter overall survival (OS) in univariate analysis. The target of homozygous deletion 1p12 was FAM46C. In addition, mutations of FAM46C were identified in 3.4% to 13% of primary MM tumors and 25% of 16 human myeloma cell lines (HMCLs), implying its potential pathogenic relevance. In other work we have suggested that FAM46C mutation is a progression event and have shown that it is rarely seen in newly diagnosed del17 patients, inferring some overlap in function. However, there is no published functional annotation of FAM46C and its role in MM remains unknown. In the present study, we aimed to identify the biological role of FAM46C in myeloma cells.Materials/Method: The expression of FAM46C in HMCLs was analyzed by western blot. Lentiviral constructs expressing wild type and mutated FAM46C were generated and transduced into HMCLs, followed by cell viability assay and cell cycle analysis. Cells were harvested and processed to measure gene expression and cell signaling changes after introduction of FAM46C by mRNAseq, pathway analysis and immunoblotting assay.Results: The expression of FAM46C protein is generally low in most HMCLs. Introduction of wild type FAM46C in HMCLs induced a substantial cell growth inhibition and apoptosis, especially in two HMCLs including MM1.S and KMS11. Cell viability of KMS11 and MM1.S was reduced by 50% to 80% at day 6 after introduction of FAM46C, compared to 0-30% growth retardation detected in HMCLs and non-myeloma cell lines that do not carry FAM46C deletion. We identified 88 genes whose mRNA expression was significantly altered after enforced expression of FAM46C in MM1.S cells. Pathway analysis revealed that FAM46C-regulated genes are enriched in the canonical pathways associated with unfolded protein response, cell cycle control and DNA damage repair. Critical MM genes that are downregulated by FAM46C expression include IRF4 and MYC, which are also downstream targets of immunomodulatory drugs (IMiDs). Consistently, some HMCLs such as KMS11 and OPM2 show an enhanced sensitivity to lenalidomide after introduction of FAM46C. Next, lentiviral constructs expressing various FAM46C mutants were generated in order to understand the consequence of FAM46C mutation. The mutant constructs mimic mutations identified in MM patients or HMCLs. Those mutants and wild type FAM46C were transduced and tested together in MM1.S cells. We found that three published misssense mutations, one frame-shift mutation and deletion of the sequence between aa172 and aa186 of FAM46C (which has been found in previous studies as a hot spot of mutation) all abolished FAM46C-mediated anti-myeloma activity, thus would be expected to confer a MM cell survival advantage.Conclusion: Our data demonstrated that enforced FAM46C expression in myeloma cells induced myeloma growth inhibition and apoptosis. Mutations in FAM46C and TP53 in newly diagnosed patients seem mutually exclusive but not in relapsed patients from our patients sequencing studies, suggesting it may associate with disease progression. Together, these studies suggest that FAM46C may function as a tumor suppressor in myeloma. We also found that published mutations of FAM46C confer a survival advantage to MM cells, and that FAM46C overexpression downregulates IRF4 and MYC and is thus associated with loss of myeloma cell survival. DisclosuresStewart:Novartis: Consultancy; Oncospire Inc.: Equity Ownership; Celgene: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees.

Next Generation XPO1 Inhibitor KPT-8602 for the Treatment of Drug-Resistant Multiple Myeloma

PurposeHuman multiple myeloma (MM) remains an incurable disease despite relatively effective treatments, including proteasome inhibitors, immunomodulator-based therapies, and high-dose chemotherapy with autologous stem cell rescue. New agents are needed to further improve treatment outcomes. In previous studies, we have shown that inhibitors of the nuclear export receptor XPO1, in combination with bortezomib, carfilzomib, doxorubicin, or melphalan, synergistically induced apoptosis in MM cells in vitro, in vivo and ex vivo without affecting non-myeloma cells. In early clinical trials, the oral, brain penetrating XPO1 inhibitor selinexor showed clear anti-myeloma activity however adverse events have been recorded, including nausea and anorexia. Our purpose was to investigate the use of oral KPT-8602, a novel small-molecule inhibitor of XPO1 with minimal brain penetration, which has been shown to have reduced toxicities in rodents and primates while maintaining potent anti-tumor effects.Experimental ProceduresTo test the efficacy of KPT-8602, we treated human MM cell lines (both parental and drug-resistant) with KPT-8602 ± currently used MM drugs, including bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, and VP-16. Human MM cell lines assayed included RPMI-8226 (8226), NCI-H929 (H929), U266, and MM1.S, PI-resistant 8226-B25 and U266-PSR, doxorubicin-resistant 8226-Dox6 and 8226-Dox40, and melphalan-resistant 8226-LR5 and U266-LR6 cell lines. MM cells (2-4x106 cells/mL) were treated for 24 hours with KPT-8602 (300 nM), followed by treatment with one of the listed anti-MM agents for an additional 24 hours. MM cells were then assayed for cell viability (CellTiter-Blue, Promega). In addition, cells were treated with KPT-8602 ± anti-MM agents concurrently for 20 hours and assayed for apoptosis by flow cytometry. In vivo testing was done in NOD/SCID-g mice by intradermal injection of U266 MM cells. Treatment started 2 weeks after tumor challenge with KPT-8602 (10 mg/kg) ± melphalan (1 or 3 mg/kg) 2X/week (Tuesday, Friday) or with KPT-8602 alone 5X weekly (10 mg/kg) (Monday-Friday). A parallel experiment was run using the clinical XPO1 inhibitor KPT-330 (selinexor). Ex vivo testing was performed on MM cells from newly diagnosed/relapsed patient bone marrow aspirates with KPT-8602 ± bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, or VP16. CD138+/light-chain+ cells were assayed for apoptosis by flow cytometry.ResultsViability assay showed that KPT-8602 had low IC50values (~140 nM) as a single agent and functioned synergistically with bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16. (CI values < 1.0). This synergistic effect was less pronounced in myeloma cells when KPT-8602 was used in combination with dexamethasone or lenalidomide. KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 combination therapy also induced apoptosis in all MM cell lines tested, including drug-resistant cell lines, as shown by caspase 3 cleavage and flow cytometric analyses. NOD/SCID-gamma mouse tumor growth was reduced and survival increased in KPT-8602/melphalan-treated mice when compared to single-agent controls. In addition, mice treated with KPT-8602 5X weekly had significantly reduced tumor growth and increased survival when compared to 2X weekly drug administration. No toxicity was observed in KPT-8602-treated mice as determined by weight loss in both the 2X and 5X groups. In patient bone marrow biopsies, the combination of KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 was more effective than single agents at inducing apoptosis in CD138+/LC+ MM cells in both newly diagnosed and relapsed/refractory patient samples.ConclusionsWe found that the novel XPO1 inhibitor KPT-8602 sensitizes MM cells to bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 as shown by apoptosis in parental and drug-resistant cell lines and by cell viability assays. Sensitization was found to be synergistic. In addition, KPT-8602 was effective in treatment of human MM tumors in mice as a single agent or in combination with melphalan and was effective when combined with several MM drugs in MM cell lines and MM patient bone marrow aspirates. KPT-8602 may be a potential candidate for future clinical trials. DisclosuresShacham:Karyopharm: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties.

Selective Inhibition Of The PI3K-Alpha Isoform Blocks Myeloma Cell Growth and Survival

IntroductionBoth, the phosphoinositide-3kinase (PI3K)/AKT pathway as well as its nutrient-dependent downstream target, the mToR (mammalian target of rapamycin) kinase, are essential for the growth and survival of malignant plasma cells. PI3K exists in four isotypes (α - δ), the role of each of these isotypes in multiple myeloma (MM), however, is not defined. Therefore, we evaluated systematically the in vitroanti-myeloma activity of NVP-BYL719 (Novartis), a newly developed inhibitor highly selective for the PI3K α isoform, and compared it with the activity of other inhibitors targeting the PI3K/AKT/mToR signaling network at different sites. Since inhibition of mToRC1 may lead to PI3K activation by a feedback loop, we looked also at combinations of NVP-BYL719 and rapalogs. Materials and MethodsAn MTS based growth assay was used to measure growth inhibition of NVP-BYL719 in comparison to different inhibitors (the pan-PI3K inhibitors Ly294002 and NVP-BKM120, the dual PI3K/mToR inhibitor NVP-BEZ235, and the allosteric mToR inhibitors rapamycin and everolimus) on six human malignant plasma cell lines and two non-myeloma cell lines, K562 and Raji. Western blot analysis was used to confirm inhibitor activity and specificity. In addition, combinations of PI3K and mToR inhibitors were tested and antagonistic/synergistic activity was calculated with the CalcuSynTM(Biosoft) software. ResultsThe selective PI3K alpha inhibitor NVP-BYL719 inhibited myeloma cell growth at a dose level (IC50 < 10 µM in 5/6 cell lines), where non-MM cell lines were not affected. Only cell line U266 was showed moderate sensitivity. The levels of the PI3K isoforms αwere comparable in all tested MM cell , as shown by Western blot. NVP-BYL719 blocked phosphorylation of AKT and of protein S6, an mToR downstream target. Of note, whenever rapamycin was combined with a PI3K inhibitor including NVP-BYL719, synergistic growth inhibition was seen, even in combination with the dual PI3K/mToR inhibitor NVP-BEZ235. ConclusionsHere, the PI3Kα isoform is reported as being essential for the growth of myeloma cells. This provides the rationale for the therapeutic exploration of such isoform specific PI3K inhibitors in MM allowing dose intensification with less toxicity. Allosteric mToR inhibitors, already active in early clinical trials in MM patients may provide interesting combination partners for these PI3K-inhibitors. Disclosures:Guenther:Novartis: Consultancy. Gramatzki:Novartis: Consultancy.

Anti-CD47 antibodies promote phagocytosis and inhibit the growth of human myeloma cells

Multiple myeloma is a plasma cell neoplasm residing in bone marrow. Despite advances in myeloma therapies, novel therapies are required to improve patient outcomes. CD47 is highly expressed on myeloma cells and a potential therapeutic candidate for myeloma therapies. Flow cytometric analysis of patient bone marrow cells revealed that myeloma cells overexpress CD47 when compared with non-myeloma cells in 73% of patients (27/37). CD47 expression protects cells from phagocytosis by transmitting an inhibitory signal to macrophages. Here we show that blocking CD47 with an anti-CD47 monoclonal antibody increased phagocytosis of myeloma cells in vitro. In xenotransplantation models, anti-CD47 antibodies inhibited the growth of RPMI 8226 myeloma cells and led to tumor regression (42/57 mice), implicating the eradication of myeloma-initiating cells. Moreover, anti-CD47 antibodies retarded the growth of patient myeloma cells and alleviated bone resorption in human bone-bearing mice. Irradiation of mice before myeloma cell xenotransplantation abolished the therapeutic efficacy of anti-CD47 antibodies delivered 2 weeks after radiation, and coincided with a reduction of myelomonocytic cells in spleen, bone marrow and liver. These results are consistent with the hypothesis that anti-CD47 blocking antibodies inhibit myeloma growth, in part, by increasing phagocytosis of myeloma cells.

Identification of Molecular Vulnerabilities in Human Multiple Myeloma Cells by RNA Interference Lethality Screening of the Druggable Genome

Despite recent advances in targeted treatments for multiple myeloma, optimal molecular therapeutic targets have yet to be identified. To functionally identify critical molecular targets, we conducted a genome-scale lethality study in multiple myeloma cells using siRNAs. We validated the top 160 lethal hits with four siRNAs per gene in three multiple myeloma cell lines and two non-myeloma cell lines, cataloging a total of 57 potent multiple myeloma survival genes. We identified the Bcl2 family member MCL1 and several 26S proteasome subunits among the most important and selective multiple myeloma survival genes. These results provided biologic validation of our screening strategy. Other essential targets included genes involved in RNA splicing, ubiquitination, transcription, translation, and mitosis. Several of the multiple myeloma survival genes, especially MCL1, TNK2, CDK11, and WBSCR22, exhibited differential expression in primary plasma cells compared with other human primary somatic tissues. Overall, the most striking differential functional vulnerabilities between multiple myeloma and non-multiple myeloma cells were found to occur within the 20S proteasome subunits, MCL1, RRM1, USP8, and CKAP5. We propose that these genes should be investigated further as potential therapeutic targets in multiple myeloma.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Use of an ex vivo multiplexed signal pathway inhibitor treatment to reveal sensitivity of myeloma and nonmyeloma bone marrow cell populations.

e18532 Background: Myeloma cells are admixed with normal cells in the bone microenvironment. A great need exists to understand the differential effect of treatment on myeloma as well as the non-myeloma cells. We designed a study to rapidly screen ex vivo treatment combinations to predict treatment efficacy. Methods: A panel of signal pathway inhibitor treatments was applied to fresh bone marrow aspirate samples from patients undergoing standard of care hematological work up for multiple myeloma. Bone marrow aspirates (n=22 to date), not required for diagnosis, were immediately subdivided and treated ex vivo with a panel of molecular targeted inhibitors, including combinations thereof, that target a wide range of cellular pathways (autophagy, proteosome, angiogenesis, protein degradation, proliferation/survival, insulin response, and translation). Up to 48 different treatment conditions could be studied for an aspirate volume of 5mL. After 8 hours incubation the samples were placed in a preservative that suppresses fluctuations in kinase pathway proteins. CD138 positive cells (plasma cells) were separated from CD138 negative cells via immunomagnetic sorting. Reverse phase protein microarrays were used to quantitatively map up to 75 cell signaling pathway endpoints in both cell populations. Results: Accrual has included untreated patients with MGUS, smoldering myeloma, treatment naïve, or advanced stage pre-treated myeloma. Individual samples and disease stages differed widely at the level of cellular signaling in response to ex vivo treatment and combination of drug treatments. In some samples specific kinase inhibitors were found to suppress their expected targets to a higher degree in CD138 positive vs CD138 negative cells. In other samples the same inhibitor was associated with compensatory activation of down stream pathways. However, in many cases the specific inhibitor had a much greater effect on the non-CD138 positive cells compared to the CD138 positive cells. Conclusions: Choices for individualized and/or combination therapy should take into consideration the differential effect of therapy on the non-myeloma cells compared to the myeloma cells. No significant financial relationships to disclose.

Kinome-Wide RNAi Studies in Human Multiple Myeloma Identify a Lymphoid Restricted Kinase GRK6 as a Selectively Vulnerable Target That Regulates STAT3/MCL1.

Abstract 601A paucity of validated kinase targets in human multiple myeloma (MM) has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in MM tumor lines bearing common t(4;14), t(14;16) and t(11;14) translocations to identify critically vulnerable kinases in MM tumor cells without regard to preconceived mechanistic notions. Primary screening was performed in duplicate using an 1800-oligo siRNA library in a single-siRNA-per-well format. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. After 96 hours, viability was measured by ATP-dependent luminescence. Fifteen kinases were consistently vulnerable in MM cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. While several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly the G-protein coupled receptor kinase, GRK6, appeared selectively vulnerable in MM. GRK6 inhibition is selectively lethal to KMS11, OPM1, H929, KMS18 and OCI-MY5 myeloma cells and has minimal effect on 293, MCF7, SF767, A375 or A549 epithelial cells. Persistent expression of FLAG-GRK6 via cDNA rescued KMS11 cells from the lethal effect of a 3'UTR-targeted GRK6 siRNA, but not from control siRNA, validating identification of GRK6 as an essential myeloma survival kinase. Furthermore, concordant results were obtained using four different exon-based GRK6 siRNA, all of which induced GRK6 silencing and similar inhibition of KMS11 proliferation and viability. Significantly, GRK6 is ubiquitously expressed in lymphoid tissues and myeloma, but by comparison appears absent or only weakly expressed in most primary human somatic tissues. From co-immunoprecipitation experiments we demonstrate that GRK6 is highly expressed in myeloma cells via direct association with the HSP90 chaperone. Inhibition of HSP90 with geldanamycin blocks GRK6 protein expression. Importantly, direct GRK6 silencing causes rapid and selective suppression of STAT3 phosphorylation that is associated with sustained reductions in total MCL1 protein levels and MCL1 phosphorylation (within 24 hours), providing a potent mechanism for the cytotoxicity of GRK6 inhibition in MM tumor cells. GF109203X is an inhibitor of both protein kinase C and of GRK6 that causes near total inhibition of these kinases in vitro at distinct concentrations of 0.1μM and 1-10μM respectively. Notably, GF109203X was substantially cytotoxic to 10/14 myeloma tumor lines at concentrations most consistent with GRK6 inhibition (5-20μM), and was selectively more cytotoxic to myeloma tumor cells than to non-myeloma cell lines (P=0.01), highlighting the potential of GRK6 as a pharmaceutical target for selective therapeutic intervention in myeloma. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. Disclosures:Perkins:MMRC: Employment. Reeder:Celgene: Research Funding; Millennium: Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

Ex Vivo Multiplexed Signal Pathway Inhibitor Treatment Reveals Differential Sensitivity of Myeloma and Non-Myeloma Bone Marrow Cell Populations.

Abstract 2860Poster Board II-836Multiple Myeloma (MM) is a heterogeneous disease, and despite significant advances in therapy it remains incurable. A major challenge facing clinicians is to determine which new agent or which combinations of agents will prove efficacious for individual patients.Interaction of MM cells with bone marrow microenvironment cells has a pathogenetic role in the disease and confers tumor cell resistance to conventional therapies. The new generation of therapies is designed to target signal transduction pathways, particularly tyrosine kinase signaling. The phosphorylation, or activation state, of kinase-driven signal networks contains important information concerning disease pathogenesis and the ongoing state of kinase-associated therapeutic targets. Reverse-phase protein microarray (RPMA) is a reproducible, high-throughput system for protein signal pathway profiling. Phosphorylation state of kinase-associated therapeutic targets provides direct information regarding the target and off-target effects of treatment.We describe herein an ex vivo study of signal pathway inhibitor treatments of fresh bone marrow aspirate samples from patients undergoing standard of care hematological work up for multiple myeloma at any stage or treatment course. Bone marrow aspirates (n=37 to date), not required for diagnosis, were immediately subdivided and treated ex vivo with a panel of molecular targeted inhibitors and combinations, and/or exogenous ligands (SCF, IGF-1), and/or cytokines (IL-6), that target a wide range of cellular pathways (e.g. proteosome, angiogenesis related, protein degradation, cell proliferation/survival, insulin response, and protein translation). Up to 48 different treatment conditions can be studied for each patient sample from a bone marrow aspirate volume of 5.0mL. The objectives were a) measure the signal pathway perturbations caused by the inhibitor/ligand treatment in individual bone marrow aspirate samples, b) compare the relative sensitivity of tumor and non tumor bone marrow cells treated in admixture under identical conditions to identify predictive/prognostic protein-based biomarkers. We specifically measured the post-translational modifications (i.e. phosphorylation) of the drug target substrates to provide direct information regarding the target and off-target effects of treatment.After incubation for 4 hours the bone marrow aspirate samples were placed in a preservative that suppresses fluctuations in kinase pathway proteins. MM CD138 positive cells were separated from the non-CD138+ bone marrow microenvironment cells via immunomagnetic sorting. RPMA was used to quantitatively map 75 cell signaling pathway endpoints in CD138+ and non-CD138+ cell populations that were treated simultaneously prior to cell sorting.To date the accrual has included untreated patients with MGUS, smoldering myeloma, treatment naïve, or advanced stage, pre-treated myeloma. Individual patients differed widely in response to ex vivo treatment and combination of drug treatments. Dexamethasone showed differential effects on the myeloma cells compared to non-myeloma cells from the same patient. For all the untreated patients studied to date, dexamethasone significantly suppressed the phosphoAKT or NF-KB endpoints, while increasing the pro-apoptotic signaling proteins such as cleaved caspases. In contrast, all the patients heavily pre-treated with dexamethasone in combination with other agents did not show any differential effects of the dexamethasone treatment on the CD138+ cells compared to the non-CD138+ cells in the bone marrow microenvironment.Many molecular targeted inhibitors induced an up-regulation of the target pathway even in combination therapies such as Rapamycin plus an IGF-1R inhibitor. Sorafinib, a molecular targeted tyrosine kinase inhibitor which blocks the MAPK (Raf/Mek/ERK) pathway, induced a compensatory up-regulation of ERK T202/Y204 in all patients except one who was in remission after treatment.Multiplexed phosphoprotein cell signaling analysis before or after treatment with ex vivo inhibitors may predict patient-specific therapeutic response and/or off target effects. Selected agents that show efficacy in ex vivo studies may be considered for further in vivo clinical studies in which signal profiling of the bone marrow cellular populations could be performed before and after molecular targeted therapy. Disclosures:No relevant conflicts of interest to declare.

Converting Tumor-specific Markers Into Reporters of Oncolytic Virus Infection

Preferential killing of transformed cells, while keeping normal cells and organs unharmed, is the main goal of cancer gene therapy. Genetically engineered trackable markers and imaging reporters enable noninvasive monitoring of transduction efficiency and pharmacokinetics of anticancer virotherapeutics. However, none of these reporters can differentiate between infection in the targeted tumors and that in the normal tissue. Thus, we constructed oncolytic measles virus (MV) armed with a human light immunoglobulin chain reporter gene for the treatment of multiple myeloma (MM). Excessive production of monoclonal immunoglobulin is a key characteristic and marker for diagnostics of MM. Once expressed in infected target cells, vector-encoded lambda protein recombines with myeloma IgG-kappa immunoglobulin creating a unique IgG-kappa/lambda. A modified immunoassay technique allows precise quantification of converted marker molecules. Only antibody producing cells were able to assemble this chimeric immunoglobulin molecule, whereas other cells secreted only free lambda light chain. Human myeloma xenografts inoculated with lambda chain expressing MV secreted converted IgG-kappa/lambda in the plasma of tumor bearing animals and elevated reporter levels correlated with response to the therapy. This is the first report of a gene therapy vector engineered to discriminate between infection in malignant and normal cells by molecular modification of a tumor-specific protein.