Single-Cell Proteomics and Tumor RNAseq Identify Novel Pathways Associated With Clofazimine Sensitivity in PI- and IMiD- Resistant Myeloma, and Putative Stem-Like Cells.

Multiple myeloma (MM) is an incurable plasma cell malignancy with dose-limiting toxicities and inter-individual variation in response/resistance to the standard-of-care/primary drugs, proteasome inhibitors (PIs), and immunomodulatory derivatives (IMiDs). Although newer therapeutic options are potentially highly efficacious, their costs outweigh the effectiveness. Previously, we have established that clofazimine (CLF) activates peroxisome proliferator-activated receptor-γ, synergizes with primary therapies, and targets cancer stem-like cells (CSCs) in drug-resistant chronic myeloid leukemia (CML) patients. In this study, we used a panel of human myeloma cell lines as in vitro model systems representing drug-sensitive, innate/refractory, and clonally-derived acquired/relapsed PI- and cereblon (CRBN)-negative IMiD-resistant myeloma and bone marrow-derived CD138+ primary myeloma cells obtained from patients as ex vivo models to demonstrate that CLF shows significant cytotoxicity against drug-resistant myeloma as single-agent and in combination with PIs and IMiDs. Next, using genome-wide transcriptome analysis (RNA-sequencing), single-cell proteomics (CyTOF; Cytometry by time-of-flight), and ingenuity pathway analysis (IPA), we identified novel pathways associated with CLF efficacy, including induction of ER stress, autophagy, mitochondrial dysfunction, oxidative phosphorylation, enhancement of downstream cascade of p65-NFkB-IRF4-Myc downregulation, and ROS-dependent apoptotic cell death in myeloma. Further, we also showed that CLF is effective in killing rare refractory subclones like side populations that have been referred to as myeloma stem-like cells. Since CLF is an FDA-approved drug and also on WHO's list of safe and effective essential medicines, it has strong potential to be rapidly re-purposed as a safe and cost-effective anti-myeloma drug.

secDrug: a pipeline to discover novel drug combinations to kill drug-resistant multiple myeloma cells using a greedy set cover algorithm and single-cell multi-omics.

Multiple myeloma, the second-most common hematopoietic malignancy in the United States, still remains an incurable disease with dose-limiting toxicities and resistance to primary drugs like proteasome inhibitors (PIs) and Immunomodulatory drugs (IMiDs).We have created a computational pipeline that uses pharmacogenomics data-driven optimization-regularization/greedy algorithm to predict novel drugs ("secDrugs") against drug-resistant myeloma. Next, we used single-cell RNA sequencing (scRNAseq) as a screening tool to predict top combination candidates based on the enrichment of target genes. For in vitro validation of secDrugs, we used a panel of human myeloma cell lines representing drug-sensitive, innate/refractory, and acquired/relapsed PI- and IMiD resistance. Next, we performed single-cell proteomics (CyTOF or Cytometry time of flight) in patient-derived bone marrow cells (ex vivo), genome-wide transcriptome analysis (bulk RNA sequencing), and functional assays like CRISPR-based gene editing to explore molecular pathways underlying secDrug efficacy and drug synergy. Finally, we developed a universally applicable R-software package for predicting novel secondary therapies in chemotherapy-resistant cancers that outputs a list of the top drug combination candidates with rank and confidence scores.Thus, using 17AAG (HSP90 inhibitor) + FK866 (NAMPT inhibitor) as proof of principle secDrugs, we established a novel pipeline to introduce several new therapeutic options for the management of PI and IMiD-resistant myeloma.

In vitro and ex vivo gene expression profiling reveals differential kinetic response of HSPs and UPR genes is associated with PI resistance in multiple myeloma.

Extensive inter-individual variation in response to chemotherapy (sensitive vs resistant tumors) is a serious cause of concern in the treatment of multiple myeloma (MM). In this study, we used human myeloma cell lines (HMCLs), and patient-derived CD138+ cells to compare kinetic changes in gene expression patterns between innate proteasome inhibitor (PI)-sensitive and PI-resistant HMCLs following test dosing with the second-generation PI Ixazomib. We found 1553 genes that changed significantly post treatment in PI-sensitive HMCLs compared with only seven in PI-resistant HMCLs (p < 0.05). Genes that were uniquely regulated in PI-resistant lines were RICTOR (activated), HNF4A, miR-16-5p (activated), MYCN (inhibited), and MYC (inhibited). Ingenuity pathway analysis (IPA) using top kinetic response genes identified the proteasome ubiquitination pathway (PUP), and nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response as top canonical pathways in Ix-sensitive cell lines and patient-derived cells, whereas EIF2 signaling and mTOR signaling pathways were unique to PI resistance. Further, 10 genes were common between our in vitro and ex vivo post-treatment kinetic PI response profiles and Shaughnessy's GEP80-postBz gene expression signature, including the high-risk PUP gene PSMD4. Notably, we found that heat shock proteins and PUP pathway genes showed significant higher upregulation in Ix-sensitive lines compared with the fold-change in Ix-resistant myelomas.

Glutaminase inhibitor CB-839 synergizes with carfilzomib in resistant multiple myeloma cells.

Curative responses in the treatment of multiple myeloma (MM) are limited by the emergence of therapeutic resistance. To address this problem, we set out to identify druggable mechanisms that convey resistance to proteasome inhibitors (PIs; e.g., bortezomib), which are cornerstone agents in the treatment of MM. In isogenic pairs of PI sensitive and resistant cells, we observed stark differences in cellular bioenergetics between the divergent phenotypes. PI resistant cells exhibited increased mitochondrial respiration driven by glutamine as the principle fuel source. To target glutamine-induced respiration in PI resistant cells, we utilized the glutaminase-1 inhibitor, CB-839. CB-839 inhibited mitochondrial respiration and was more cytotoxic in PI resistant cells as a single agent. Furthermore, we found that CB-839 synergistically enhanced the activity of multiple PIs with the most dramatic synergy being observed with carfilzomib (Crflz), which was confirmed in a panel of genetically diverse PI sensitive and resistant MM cells. Mechanistically, CB-839 enhanced Crflz-induced ER stress and apoptosis, characterized by a robust induction of ATF4 and CHOP and the activation of caspases. Our findings suggest that the acquisition of PI resistance involves adaptations in cellular bioenergetics, supporting the combination of CB-839 with Crflz for the treatment of refractory MM.

Phenotypic and functional characterization of a bortezomib-resistant multiple myeloma cell line by flow and mass cytometry.

Multiple myeloma (MM) is an incurable malignant plasma cell neoplasm. Proteasome inhibitors including Bortezomib (Bz) are used to treat MM, and treatment failure due to drug resistance occurs. Bz-sensitive and -resistant MM cells have distinct immunophenotypic signatures that correlate with clinical outcome. These changes can be identified by fluorescence-based cytometry (FBC), however, FBC is rarely used in predicting Bz resistance. Mass cytometry (MC) is a recently developed variation of flow cytometry that detects heavy metal-ion tagged antibodies using time-of-flight mass spectrometry allowing for detection of up to 38 epitopes simultaneously in a single cell, without significant overlap, exceeding the dimensionality of FBC 3-4-fold. Here, we compared FBC and MC in the immunophenotypic characterization of Bz-sensitive and -resistant human MM cell line U266. We show that Bz-resistant cells are associated with the loss of CD56 and CD66a adhesion molecules as well as an activation signature.

Fine-Mapping of 18q21.1 Locus Identifies Single Nucleotide Polymorphisms Associated with Nonsyndromic Cleft Lip with or without Cleft Palate.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common congenital birth defects. NSCL/P is a complex multifactorial disease caused by interactions between multiple environmental and genetic factors. However, the causal single nucleotide polymorphism (SNP) signature profile underlying the risk of familial NSCL/P still remains unknown. We previously reported a 5.7-Mb genomic region on chromosome 18q21.1 locus that potentially contributes to autosomal dominant, low-penetrance inheritance of NSCL/P. In the current study, we performed exome sequencing on 12 familial genomes (six affected individuals, two obligate carriers, and four seemingly unaffected individuals) of a six-generation family to identify candidate SNPs associated with NSCL/P risk. Subsequently, targeted bidirectional DNA re-sequencing of polymerase chain reaction (PCR)-amplified high-risk regions of MYO5B gene and sequenom iPLEX genotpying of 29 candidate SNPs were performed on a larger set of 33 members of this NSCL/P family (10 affected + 4 obligate carriers + 19 unaffected relatives) to find SNPs significantly associated with NSCL/P trait. SNP vs. NSCL/P association analysis showed the MYO5B SNP rs183559995 GA genotype had an odds ratio of 18.09 (95% Confidence Interval = 1.86-176.34; gender-adjusted P = 0.0019) compared to the reference GG genotype. Additionally, the following SNPs were also found significantly associated with NSCL/P risk: rs1450425 (LOXHD1), rs6507992 (SKA1), rs78950893 (SMAD7), rs8097060, rs17713847 (SCARNA17), rs6507872 (CTIF), rs8091995 (CTIF), and rs17715416 (MYO5B). We could thus identify mutations in several genes as key candidate SNPs associated with the risk of NSCL/P in this large multi-generation family.

PIAS1 Promotes Lymphomagenesis through MYC Upregulation.

The MYC proto-oncogene is a transcription factor implicated in a broad range of cancers. MYC is regulated by several post-translational modifications including SUMOylation, but the functional impact of this post-translational modification is still unclear. Here, we report that the SUMO E3 ligase PIAS1 SUMOylates MYC. We demonstrate that PIAS1 promotes, in a SUMOylation-dependent manner, MYC phosphorylation at serine 62 and dephosphorylation at threonine 58. These events reduce the MYC turnover, leading to increased transcriptional activity. Furthermore, we find that MYC is SUMOylated in primary B cell lymphomas and that PIAS1 is required for the viability of MYC-dependent B cell lymphoma cells as well as several cancer cell lines of epithelial origin. Finally, Pias1-null mice display endothelial defects reminiscent of Myc-null mice. Taken together, these results indicate that PIAS1 is a positive regulator of MYC.

Utilization of translational bioinformatics to identify novel biomarkers of bortezomib resistance in multiple myeloma.

Multiple myeloma (MM) is an incurable malignant neoplasm hallmarked by a clonal expansion of plasma cells, the presence of a monoclonal protein in the serum and/or urine (M-spike), lytic bone lesions, and end organ damage. Clinical outcomes for patients with MM have improved greatly over the last decade as a result of the re-purposing of compounds such as thalidomide derivatives, as well as the development of novel chemotherapeutic agents including first and second generation proteasome inhibitors, bortezomib (Bz) and carfilzomib. Unfortunately, despite these improvements, the majority of patients relapse following treatment. While Bz, one of the most commonly used proteasome inhibitors, has been successfully incorporated into clinical practice, some MM patients have de novo resistance to Bz, and the majority of the remainder subsequently develop drug resistance following treatment. A significant gap in clinical care is the lack of a reliable clinical test that would predict which MM patients have or will subsequently develop Bz resistance. Thus, as Bz resistance remains a significant challenge, research efforts are needed to identify novel biomarkers of early Bz resistance, particularly when an early therapeutic intervention can be initiated. Recent advances in MM research indicate that genomic data can be extracted to identify novel biomarkers that can be utilized to select more effective, personalized treatment protocols for individual patients. Computationally integrating large patient databases with data from whole transcriptome profiling and laboratory-based models can potentially revolutionize our understanding of MM disease mechanisms. This systems-wide approach can provide rational therapeutic targets and novel biomarkers of risk and treatment response. In this review, we discuss the use of high-content datasets (predominantly gene expression profiling) to identify novel biomarkers of treatment response and resistance to Bz in MM.

Profiling bortezomib resistance identifies secondary therapies in a mouse myeloma model.

Multiple myeloma is a hematologic malignancy characterized by the proliferation of neoplastic plasma cells in the bone marrow. Although the first-to-market proteasome inhibitor bortezomib (Velcade) has been successfully used to treat patients with myeloma, drug resistance remains an emerging problem. In this study, we identify signatures of bortezomib sensitivity and resistance by gene expression profiling (GEP) using pairs of bortezomib-sensitive (BzS) and bortezomib-resistant (BzR) cell lines created from the Bcl-XL/Myc double-transgenic mouse model of multiple myeloma. Notably, these BzR cell lines show cross-resistance to the next-generation proteasome inhibitors, MLN2238 and carfilzomib (Kyprolis) but not to other antimyeloma drugs. We further characterized the response to bortezomib using the Connectivity Map database, revealing a differential response between these cell lines to histone deacetylase (HDAC) inhibitors. Furthermore, in vivo experiments using the HDAC inhibitor panobinostat confirmed that the predicted responder showed increased sensitivity to HDAC inhibitors in the BzR line. These findings show that GEP may be used to document bortezomib resistance in myeloma cells and predict individual sensitivity to other drug classes. Finally, these data reveal complex heterogeneity within multiple myeloma and suggest that resistance to one drug class reprograms resistant clones for increased sensitivity to a distinct class of drugs. This study represents an important next step in translating pharmacogenomic profiling and may be useful for understanding personalized pharmacotherapy for patients with multiple myeloma.

Targeted overexpression of an activated N-ras gene results in B-cell and plasma cell lymphoproliferation and cooperates with c-myc to induce fatal B-cell neoplasia.

Multiple myeloma is an incurable malignant expansion of plasma cells in the bone marrow. Although there is no pathognomonic genetic lesion among multiple myeloma patients, activation of the ras gene has been identified as a common mutation. We have previously described the use of the 3' κ immunoglobulin light chain enhancer (3'KE) to target transgenic expression in murine B and plasma cells, resulting in bcl-X(L) and c-myc-driven murine models of multiple myeloma. In this report, we characterize the role of activated mutant N-ras in B and plasma cells in transgenic mice. We constructed transgenic mice that use 3'KE to direct expression of a mutant activated N-ras. We also crossed the N-ras mice with mice bearing a c-myc transgene to study the cooperative effects of the transgenic constructs. Mice were sacrificed when moribund or at specific time intervals and characterized by serology, light microscopy, and flow cytometry. The transgenic N-ras animals develop B- and plasma cell lymphoproliferation, and aged mice develop immunoglobulinemia, renal hyaline tubular casts, and microscopic foci of abnormal plasma cells in extramedullary sites, including the liver and kidney. Bitransgenic 3'KE/N-Ras V12 × Eµ-c-Myc mice develop fatal B-cell neoplasia, with a median survival of 10 weeks. These data indicate that activated N-ras can play a role in B- and plasma cell homeostasis and that activated N-Ras and c-Myc can cooperate to induce B-cell neoplasia.

A transgenic mouse model of plasma cell malignancy shows phenotypic, cytogenetic, and gene expression heterogeneity similar to human multiple myeloma.

Multiple myeloma is an incurable plasma cell malignancy for which existing animal models are limited. We have previously shown that the targeted expression of the transgenes c-Myc and Bcl-X(L) in murine plasma cells produces malignancy that displays features of human myeloma, such as localization of tumor cells to the bone marrow and lytic bone lesions. We have isolated and characterized in vitro cultures and adoptive transfers of tumors from Bcl-xl/Myc transgenic mice. Tumors have a plasmablastic morphology and variable expression of CD138, CD45, CD38, and CD19. Spectral karyotyping analysis of metaphase chromosomes from primary tumor cell cultures shows that the Bcl-xl/Myc tumors contain a variety of chromosomal abnormalities, including trisomies, translocations, and deletions. The most frequently aberrant chromosomes are 12 and 16. Three sites for recurring translocations were also identified on chromosomes 4D, 12F, and 16C. Gene expression profiling was used to identify differences in gene expression between tumor cells and normal plasma cells (NPC) and to cluster the tumors into two groups (tumor groups C and D), with distinct gene expression profiles. Four hundred and ninety-five genes were significantly different between both tumor groups and NPCs, whereas 124 genes were uniquely different from NPCs in tumor group C and 204 genes were uniquely different from NPCs in tumor group D. Similar to human myeloma, the cyclin D genes are differentially dysregulated in the mouse tumor groups. These data suggest the Bcl-xl/Myc tumors are similar to a subset of plasmablastic human myelomas and provide insight into the specific genes and pathways underlying the human disease.

Gene profiling of a myeloma cell line reveals similarities and unique signatures among IL-6 response, N-ras-activating mutations, and coculture with bone marrow stromal cells.

ANBL-6, a myeloma cell line, proliferates in response to interleukin 6 (IL-6) stimulation, coculture with bone marrow stromal cells, and when harboring a constitutively active mutant N-ras gene. Eighteen samples, including 4 IL-6-treated, 3 mutant N-ras-transfected, 3 normal stroma-stimulated, 2 multiple myeloma (MM) stroma-stimulated, and 6 untreated controls were profiled using microarrays interrogating 12 626 genes. Global hierarchical clustering analysis distinguished at least 6 unique expression signatures. Notably, the different stimuli altered distinct functional gene programs. Class comparison analysis (P =.001) revealed 138 genes (54% involved in cell cycle) that distinguished IL-6-stimulated versus nontreated samples. Eighty-seven genes distinguished stroma-stimulated versus IL-6-treated samples (22% encoded for extracellular matrix [ECM] proteins). A total of 130 genes distinguished N-ras transfectants versus IL-6-treated samples (26% involved in metabolism). A total of 157 genes, 20% of these involved in signaling, distinguished N-ras from stroma-interacting samples. All 3 stimuli shared 347 genes, mostly of metabolic function. Genes that distinguished MM1 from MM4 clinical groups were induced at least by one treatment. Notably, only 3 genes (ETV5, DUSP6, and KIAA0735) are uniquely induced in mutant ras-containing cells. We have demonstrated gene expression patterns in myeloma cells that distinguish an intrinsic genetic transformation event and patterns derived from both soluble factors and cell contacts in the bone marrow microenvironment.