CRISPR Activation Screen for Drivers of MM Cell Proliferation

Abstract CRISPR/Cas9-based gene editing has become a powerful tool for loss-of-function (LOF) studies and has allowed us to systematically interrogate the function of genes regulating the survival and proliferation of multiple myeloma (MM) cells in vitro, in vivo and in the context of treatment resistance (e.g. De Matos Simoes et al., Shirasaki et al., and Gandolfi et al. ASH 2017). We reasoned, however, that important additional information can be obtained from CRISPR-based gain-of-function (GOF) approaches which can achieve transcriptional activation at endogenous genomic loci. We thus performed genome-scale CRISPR activation studies using the dCas9-P65-HSF transcriptional activation system (in which a Cas9 variant lacking nuclease activity [dCas9] confers P65-HSF-mediated activation of genes recognized by sgRNAs against their promoter regions). Specifically, MM.1S cells were transduced with the dCas9-P65-HSF system and pooled lentiviral particles of the Calabrese CRISPR activation sgRNA library, consisting of 2 pooled sgRNA sub-libraries (total of ~110,000 sgRNAs targeting ~18000 genes, at initial coverage of 800 cells/sgRNA). Cells were cultured for 12 weeks and harvested at baseline and various intervals, e.g. 4 and 12 weeks of culture. Next generation sequencing of genomic DNA quantified the abundance of sgRNAs in the tumor cell population and genes were ranked (with rank aggregation algorithms) in terms of their sgRNA enrichment or depletion. These analyses allowed us to observe a series of genes with statistically significant sgRNA enrichment and known or presumed roles in MM biology, including key MM transcription factors such as IRF4, the thalidomide derivative targets IKZF3 and IKZF1, and the co-factor POU2AF1; known oncogenes, e.g. KRAS and MYC; NF-kappaB pathway members, e.g. RELA; and signal transduction regulators, e.g. IGF1R and its downstream effectors IRS1 and AKT2. These results are consistent with the major depletion of sgRNAs for these genes in loss-of-function (LOF) CRISPR knockout studies. However, several other genes with significant sgRNA enrichment in CRISPR activation studies did not exhibit major sgRNA depletion in CRISPR knockout studies, including the B/plasma cell transcription factor POU2F2 (Oct2), for which high protein expression correlates with reduced survival in MM (Toman I. et al 2011); the transcription factor PAX2, the TRAF interacting protein TIFA, or the Toll-like receptor TLR4. Interestingly, significant depletion of sgRNAs was observed for several genes with known or proposed tumor suppressive properties including YAP1 (an oncogene for solid tumors, but reported as tumor suppressor for MM and other blood cancers); the pro-death TNFRSF10A (TRAIL receptor DR4), TP73, CDKN1A, the negative regulator of c-Myc MXI1, or the pro-apoptotic Bcl2 family member BAK1. Depletion or enrichment of sgRNAs for most of the aforementioned genes was detectable by 4 weeks of culture, while more pronounced changes and detection of additional statistically-significant hits was observed in later time-points. For genes with significant sgRNA enrichment in our CRISPR activation study, we examined a series of molecular alterations, including transcript overexpression in MM cell lines or patient-derived samples vs. normal plasma cells, or relapsed/refractory MM vs. earlier disease MM stages; mutational status; correlation of transcript levels with clinical outcome in MM; and extent of open chromatin (based on H3K27Ac chromatin marks) within or proximal to each gene in MM cell lines. Some "hits" from our screen exhibited at least one of these molecular alterations, but most genes harbored no such alterations or their magnitude or frequency ranked outside the top 50-100 genes. These results suggest that CRISPR activation studies can identify important putative regulators of MM biology, which may not be readily detectable based on known annotations of the MM cell genome, transcriptome, or epigenome. Genome-scale CRISPR-based transcriptional activation are an important gain-of-function system to uncover genes which induce vs. suppress tumor cell survival and proliferation, and provide information orthogonal to those yielded by other CRISPR-based approaches that involve loss-of-function interventions. Our use of CRISPR activation allowed us to both validate previously known genes and identify promising new candidate regulators of MM cell biology. Disclosures Mitsiades: EMD Serono: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/ Johnson & Johnson: Research Funding.

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Functional Genomic Landscape of Genes with Recurrent Mutations in Multiple Myeloma

Blood ◽

10.1182/blood-2018-189 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 189-189 ◽

Cited By ~ 1

Author(s):

Ricardo De Matos Simoes ◽

Ryosuke Shirasaki ◽

Huihui Tang ◽

Michal Sheffer ◽

Olga Dashevsky ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Cell ◽

Cell Survival ◽

Cell Lines ◽

Research Funding ◽

Functional Genomic ◽

Loss Of Function ◽

Functional Studies ◽

Tumor Cell Survival ◽

Genome Scale

Abstract During the last two decades, cell lines and patient-derived samples from multiple myeloma (MM) have been extensively profiled for alterations in their genome with the anticipation that those genes with the most recurrent lesions could represent attractive novel therapeutic targets or markers for aggressive disease. Yet for many of these genes, their functional significance for MM cells has not been formally evaluated. With the advent of new CRISPR/Cas9-based functional genomics platforms, it is possible to generate in genome- or subgenome-scale direct quantitative information on the impact that perturbation of these genes exerts on tumor cell survival, proliferation or other phenotypes. We therefore examined the landscape of our CRISPR-based functional genomic data for these recurrently dysregulated genes We specifically curated information from the MMRF CoMMpass study and multiple other publicly available studies, to identify genes which are recurrently identified to harbor nonsynonymous mutation (SNV or indel), DNA copy number loss or gain, or participation in chromosomal translocations. We then examined the patterns of results for these genes in our genome scale CRISPR-based gene-editing studies for loss-of-function in n=18 MM cell lines. We identified a subset of genes (e.g. FAM46C, CDKN2C, RASA2) which are considered targets for recurrent loss-of-function events and indeed exhibit, for large fractions of the cell lines tested enrichment, of their sgRNAs in CRISPR knock-out studies, consistent with a role of these genes as suppressors of tumor cell survival or proliferation. CRISPR KO of TP53 leads to increased survival/proliferation of only a small minority (2/18 of cell lines tested thus far), which reflects the fact that the overwhelming majority of MM cell lines already harbor LOF events for this gene. Interestingly, a substantial number of genes which have been considered to harbor recurrent LOF events in MM patient samples (e.g. NF1, NF2, CYLD) do not exhibit sgRNA enrichment in CRISPR KO screens in the MM cell lines tested so far. In addition, several other recurrently mutated genes for which their loss- or gain-of-function status had not been previously evaluated with extensive functional studies in MM (e.g. SP140, LTB, EGR1, ATM, PARK2, PRKD2, RAPGEF5, DOCK5, TGDS, TNFAIP8) exhibit in the majority of cell lines tested in in CRISPR knockout studies no significant enrichment or depletion of their sgRNAs. In contrast, PTPN11, CREBBP, EP300, KMT2B, KMT2C, SETD2, SF3B1 and UBR5, are notable examples of recurrently mutated genes which represent dependencies for large fractions of MM cell lines in vitro. These results highlight the value of interpreting results from next generation sequencing studies in the context of information provided by the genome scale by use of functional genomic characterization of available cell line models. We envision that, similar sub-genome scale assays were performed at the level of patient derived samples will also provide direct information about the relevance of some of these genes. In addition, functional studies conducted with context of tumor-microenvironemtn compartment interactions and tumor interface will be needed to evaluate several genes identified in the study. Disclosures Licht: Celgene: Research Funding. Mitsiades:Takeda: Other: employment of a relative; Janssen/ Johnson & Johnson: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding; TEVA: Research Funding.

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Functional Characterization of Genes Driving Enhanced Biological Aggressiveness of Myeloma Cells: Identification of Novel and Understudied "Drivers" of Myeloma

Blood ◽

10.1182/blood-2019-128919 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 313-313

Author(s):

Huihui Tang ◽

Ryosuke Shirasaki ◽

Ricardo De Matos Simoes ◽

Sara Gandolfi ◽

Olga Dashevsky ◽

...

Keyword(s):

Cell Growth ◽

Cell Survival ◽

Cell Lines ◽

Transcriptional Activation ◽

Research Funding ◽

Oncology Research ◽

Positive Regulators ◽

Genome Scale ◽

The Impact ◽

Crispr Activation

Loss-of-function (LOF) studies (e.g. using RNAi or CRISPR) have historically been the main functional approaches to identify and study genes which drive the biology of Multiple Myeloma(MM) or other neoplasias. We hypothesized though that substantial complementary data can be derived from systematically examining the impact of transcriptional activation of endogenous genomic loci of different genes. To perform such gain-of-function (GOF) studies in an open-ended genome-scale manner, we applied CRISPR activation approaches in 4 MM lines (MM1S, KMS11, LP1, L363) which were transduced with the dCas9-P65-HSF transcriptional activation system and the Calabrese genome-scale sgRNA library (2 pooled sub-libraries; total of ~110,000 sgRNAs targeting promoters of ~18000 genes; initial coverage ~800-1000 cells/sgRNA). Next generation sequencing quantified the sgRNA abundance in MM cells at baseline and various time points (e.g. 2-12 weeks of culture), and rank aggregation algorithms identified genes with statistically significant enrichment or depletion of their sgRNAs, reflecting positive vs. negative, respectively, effects of GOF of these genes on MM cell survival/proliferation. These studies identified distinct groups of genes which serve as positive regulators of MM cell growth in at least 3 of the cell lines tested, including critical transcription factors (TFs)/cofactors, such as POU2F2, POU2AF1, IRF4, MYC; growth factor signaling mediators, such as IGF1R and IRS1; Ras family members (e.g. KRAS in MM1S cells); diverse members of the solute carrier family of transporters for amino acids and other bioactive small molecules. We also observed positive regulators of MM cell growth with distinct roles in certain cell lines, e.g. induction of IKZF3, IKZF1, RELA, CD48, MBNL1, PAX2 exhibited significantly more pronounced role as positive regulators of MM1S cells compared to the other cell lines tested. Notably, several positive regulators of MM growth identified in these CRISPR activation studies are not essential for MM cell survival/proliferation in CRISPR knockout studies in the same MM cell lines or many others. Examples of such genes include the TFs POU2F2 (Oct2) and PAX2, the TRAF interacting protein TIFA or the Toll-like receptor TLR4. We went on to validate these results for several such genes (e.g. POU2F2, POU2AF1) with individual sgRNAs for CRISPR activation and/or cDNA overexpression (vs. isogenic controls) in competition experiments and cell cycle analyses. We focused on further probing the mechanistic basis for the role of POU2AF1 as one of the top positive regulators of MM cell growth and performed RNA sequencing analyses of LP1 cells harboring induction of POU2AF1 expression using CRISPR activation. We observed upregulation of a distinct cluster of >50 genes that are themselves positive regulators of LP1 cell growth in our genome-scale CRISPR activation studies. While some of these are known drivers of tumor cell growth (e.g. AURKB, E2F1, FGFR3), the large majority of these POU2AF1-driven positive regulators of MM cell growth have not been previously studied in MM; and exhibit a distinct enrichment for transcriptional regulators and other genes which are not required for LP1 cell survival/proliferation in CRISPR KO studies of LP1 cells. These results indicate that CRISPR-based activation of endogenous gene expression provides data which complementary to those derived from CRISPR LOF studies. Indeed, we not only validated through our GOF studies the relevance of genes which with prominent essentiality in CRISPR LOF studies (e.g. IRF4, POU2AF1, etc.), but also identified promising new candidate regulators of MM cell biology which are not essential for baseline survival/growth of MM cells, but can induce MM cell growth when further activated. These results provide novel insights into the biology of MM cells and may have therapeutic implications towards suppressing the ability of MM cells to transition to states of advanced biological aggressiveness. Disclosures Mitsiades: Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.

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RUNX1 Is Required For Maintenance Of Established T-ALL Blasts

Blood ◽

10.1182/blood.v122.21.3742.3742 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3742-3742

Author(s):

Christopher R Jenkins ◽

Olena O Shevchuk ◽

Hongfang Wang ◽

Vincenzo Giambra ◽

Samuel D Gusscott ◽

...

Keyword(s):

T Cell ◽

Cell Growth ◽

Cell Lines ◽

Transcriptional Activation ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Dominant Negative ◽

Brdu Incorporation ◽

Loss Of Function ◽

Cell Counts

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a clinically aggressive malignancy of immature T cells. Intensive multiagent chemotherapy achieves cure in 80-90% of pediatric patients, but only 40% of adult patients survive beyond 5 years. Data from recent ChIP-seq studies has shown that RUNX1 binds throughout the T-ALL genome at sites co-occupied by known oncogenic transcription factors including TAL1 and NOTCH1. For this reason, it has been suggested that RUNX1 may be part of a transcriptional activation complex that drives an oncogenic gene expression program in this cell context. In contrast, next generation sequencing studies have recently identified heterozygous point mutations throughout the RUNX1 coding region in T-ALL including some which are predicted to encode truncated polypeptides resembling dominant negative alleles, thus raising the possibility that RUNX1 may function rather as a tumor suppressor in this context. In an effort to explore the functional role of RUNX1 in T-ALL, we examined the effect of RUNX1 knockdown in a broad panel of established human T-ALL cell lines and xenograft-expanded patient biopsy samples. Cells transduced with lentiviral shRNAs targeting coding and 3’ UTR regions of RUNX1 showed a clear growth disadvantage as compared to either non-transduced cells in the same culture or cells transduced with non-silencing shRNAs in parallel cultures. As well, absolute cell counts of cultures containing only shRUNX1-transduced cells demonstrated dramatically reduced growth rates as compared to either non-transduced or non-silencing shRNA-transduced controls. BrdU incorporation and CFSE dye dilution studies showed that most cell lines exhibited reduced proliferation in response to RUNX1 knock-down, while a subset of lines also showed reduced cell viability. These phenotypes were largely consistent across a panel of over 20 T-ALL cell lines and 4 xenograft-expanded patient samples, including several which harbored either nonsense or missense RUNX1 mutations. These results support the notion that established T-ALL cells are generally dependent on RUNX1 for continued cell growth and, in some cases, also for survival. We also explored candidate RUNX1 target genes which might be responsible for mediating the observed growth/survival phenotypes. Assembling a short list of the “usual suspects” including genes known to regulate growth of T-ALL cells generally (c-MYC, PTEN), to possess a substantial RUNX1 ChIP-seq peak in T-ALL cells (IGF1R, IL7R), or to be bona fide RUNX1 targets in other cell contexts (p21/WAF1, p27/KIP1), we performed western blot or flow cytometric analyses of multiple shRUNX1-transduced cell lines. We noted consistent regulation of some, but not all of these in a manner consistent with RUNX1 positively supporting cell growth. From these results, we conclude that RUNX1 plays a pro-oncogenic role in established T-ALL cells. We surmise that the complement of presumed loss-of-function RUNX1 mutations observed in patient T-ALLs may be indicative of its known roles in regulating normal T cell development such that loss-of-function mutations may lead to differentiation arrest and consequently promote tumor initiation. For the majority of T-ALLs that express wild-type RUNX1 proteins, however, our results suggest that RUNX1 acts functionally to support maintenance of the malignant clone by promoting expression of known oncogenic factors and repressing expression known tumor suppressors. Disclosures: Aster: Cell Signaling Technology: Consultancy; Merck, Inc.: Research Funding; Pfizer, Inc.: Research Funding; Genentech, Inc.: Honoraria.

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TRAF3 Loss Drives Alternative NF-κB Pathway Activation in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2020-141126 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Michael Y. Li ◽

Lauren C. Chong ◽

Elizabeth Chavez ◽

Bruce W Woolcock ◽

Adele Telenius ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

B Cell ◽

Cell Lines ◽

Copy Number ◽

Research Funding ◽

B Cell Lymphoma ◽

Luciferase Reporter ◽

Published Data ◽

Loss Of Function

Introduction: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor family that regulates gene expression programs contributing to inflammation and cell survival. NF-κB signaling occurs via two branches: classical and alternative, and is often enriched in somatic mutations of key pathway members in several lymphoid malignancies. Here, we reveal deregulation and constitutive activation of the alternative NF-κB pathway in a subset of DLBCL patients with recurrent genomic loss of the gene encoding tumor necrosis factor receptor-associated factor 3 (TRAF3), a regulator of the NF-κB signaling pathway. Methods and Results: To uncover novel driver mutations of DLBCL pathogenesis and tumor maintenance, we performed Affymetrix SNP6.0 copy number analysis on 347 de novo DLBCL samples from patients uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). We observed frequent, focal genomic loss of chr:14q32.31-32 which included TRAF3 and RCOR1 (7%, 22/313) in the minimally deleted region and an enrichment of activated B-cell-like (ABC) subtype cases over germinal center B-cell-like (GCB) subtype cases, confirming previously published data (Chan et al, Blood 2014). RNAseq of these DLBCL samples revealed a significant reduction of TRAF3 mRNA in chr:14q32.31-32 deleted cases compared to copy number neutral cases (p=0.002). Next, we focused on characterizing the phenotypic consequences of TRAF3 loss in DLBCL. We used CRISPR/Cas9 gene editing to knock out TRAF3 in 2 GCB-DLBCL (DOHH2, OCI-LY1) and 2 ABC-DLBCL (HBL1, OCI-LY3) cell lines. We performed immunoblotting analysis of NF-κB pathway members on cell fractionated samples of TRAF3 knockout cells and found increased levels of the NF-κB inducing kinase NIK (a direct target of TRAF3-mediated ubiquitin-proteasome degradation) and a concomitant increased nuclear translocation of NF-κB transcription factor complex subunits RelB and p52. Proteasome blockade restored RelB cytoplasmic localization and reduced processed p52 protein in TRAF3 knockout GCB-DLBCL lines only, indicating other factors may contribute to alternative NF-κB activation in ABC-DLBCL. Moreover, classical NF-κB activation remained unaffected, highlighting the specific role of TRAF3 regulation on the alternative NF-κB pathway in DLBCL. Consistent with these findings, TRAF3 knockout cells exhibited NF-κB-dependent transcriptional upregulation by luciferase reporter activity and elevated pro-inflammatory cytokine production (IL-6, TNF-β) by Luminex and ELISA. To study transcriptome changes as a result of TRAF3 loss-of-function, we performed RNAseq and differential gene expression analysis on wildtype and TRAF3 knockout DLBCL cell lines as well as primary DLBCL samples (N=347). We found enrichment of NIK and NF-κB associated pathways in TRAF3 deficient DLBCL and uncovered additional enriched gene sets including those involved in cell cycle regulation, cell division and metabolism, suggesting a potential proliferative and survival advantage. Conclusion: Our findings link TRAF3 loss-of-function to clinical and gene expression phenotypes in DLBCL and highlight alternative NF-κB activation as a pathogenically important pathway in both GCB and ABC subtypes. Future studies will be directed towards comprehensive evaluation of NF-κB inhibitors for effective blockade of constitutive alternative NF-κB activation in DLBCL. Disclosures Scott: NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; AbbVie: Consultancy.

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POU2AF1 As a Master Regulator of Oncogenic Transcription Factor Networks in Myeloma

Blood ◽

10.1182/blood-2020-142580 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-19

Author(s):

Ricardo De Matos Simoes ◽

Ryosuke Shirasaki ◽

Huihui Tang ◽

Shizuka Yamano ◽

Benjamin G Barwick ◽

...

Keyword(s):

Cell Growth ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Rna Seq ◽

Advisory Committees ◽

Genome Scale ◽

Crispr Activation

Background: Functional genomics studies based on CRISPR and shRNA have documented that multiple myeloma (MM) cells are preferentially dependent (compared to other neoplasias) on a series of TFs, including IKZF1 and IKZF3 (which are targeted by thalidomide derivatives) and others that are not amenable to degradation or small molecule inhibition. Transcriptional co-factors have been therapeutically targeted, for example, inhibitors of BRD4, a co-factor for pTEFB, can be used to down-regulate c-myc. Aim: To identify new transcriptional vulnerabilities in MM with an emphasis on transcriptional co-factors Methods: We integrated results from genome-scale studies using the AVANA library for loss-of-function by gene editing (in 19 MM lines) and the Calabrese library for CRISPR-mediated gene activation (in 5 MM lines) to identify critical transcriptional co-factors (co-TFs). RNA-Seq analysis was used to identify critical pathways affected by POU2AF1 activation and existing ChIP-Seq tracks in MM cells were reanalyzed. Results: POU2AF1 (OCA-B) was the most preferentially essential TF co-factor in MM cell lines vs. non-MM and one of top genes which, upon CRISPR activation in genome-scale studies, increased MM cell fitness in vitro. We further confirmed the role of this gene using focused libraries of sgRNAs against POU2AF1 in vitro and in an in vivo model of MM cell growth in bone marrow-like scaffolds "functionalized" with humanized mesenchymal bone marrow stromal cells to simulate the human BM. CRISPR activation of POU2AF1 is associated with increased MM cell growth. RNA-Seq of POU2AF1 activation in LP1 cells a transcriptional program characterized by upregulation of other genes that are preferentially essential for MM including PRDM1, SUPT7L, UBE2G2 and TSC1; broad-spectrum oncogenic dependencies (e.g KRAS) and genes known or proposed to be involved in the pathophysiology of MM or other neoplasias (e.g. RUNX2, FGFR3, SMO, CREB5, TNFRSF13B, MEF2D, PCGF2). POU2AF1 overexpression was also associated with down-regulation of CDKN1C; of MHC class II molecules and their transcriptional activator CIITA, suggesting that POU2AF1 activation could also contribute to increased MM growth in vivo by allowing escape from immune surveillance. ATAC-Seq data and genome-wide ChIPseq for H3K27Ac in MM cell lines indicate that chromatin surrounding the POU2AF1 locus was highly accessible, concordant with the consistent expression of this TF in MM cell lines and patient-derived cells. CoMMpass data showed that POU2AF1 expression was enhanced in a subset of MM patients at relapse compared to diagnosis. Motif analysis of ChIP-seq data for POU2AF1 identified significant overlap with motifs for TFs relevant to the POU family (e.g. Oct11, Oct2, Oct4); members of the ETS family (e.g. ELF1, Elf4, GABPA); and other TFs with roles in MM including c-myc; IRF4; NF-kappaB, PRDM1, RUNX2 and the POU2AF1 target CREB5. These data suggest a functional interaction between POU2AF1 and other MM-relevant TFs. The transcriptional signature of POU2AF1 activation is enriched for genes downregulated by suppression/inhibition of MM-preferential TFs or epigenetic regulators including IRF4, PRDM1, IKZF1/3 and DOT1L. POU2AF1 binding motifs are also enriched in the promoter regions of MM-preferential dependencies including several MM-preferential TFs. Conclusions: POU2AF1 is essential for MM cells in vitro and in vivo; has a significantly more pronounced and recurrent role as a dependency in MM compared to most other neoplasias; and can further drive MM cell growth, through its ability to interact with several TFs critical for MM, forming multi-protein functional complexes. These results establish POU2AF1 as a central component in the regulatory network of oncogenic TFs in MM and highlight the value of further exploring POU2AF1 as a therapeutic target in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Cedilla Therapeutics: Consultancy; Tango Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Karyopharm: Research Funding; TEVA: Research Funding; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding.

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Genome-scale CRISPR activation screen uncovers tumor-intrinsic modulators of CD3 bispecific antibody efficacy

Scientific Reports ◽

10.1038/s41598-019-56670-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Corinne E. Decker ◽

Tara Young ◽

Elizabeth Pasnikowski ◽

Joyce Chiu ◽

Hang Song ◽

...

Keyword(s):

T Cells ◽

Tumor Cell ◽

Cell Lines ◽

Cell Lysis ◽

Lymphoma Cell ◽

B Lymphoma ◽

Cell Clustering ◽

A Genome ◽

Genome Scale ◽

Crispr Activation

AbstractBispecific antibodies (bsAb) that bridge tumor cells and CD3-positive effector T cells are being developed against many tumor cell targets. While tumor cell factors other than target expression level appear to play a role in determining the efficacy of CD3 bsAb, the identity of such factors remains largely unknown. Using a co-culture system of primary human T cells and B lymphoma cell lines, we demonstrate a range of sensitivities to CD20xCD3 bsAb that is independent of CD20 surface expression. To identify genes that modulate tumor cell sensitivity to CD3 bsAb, we employed a genome-scale CRISPR activation screen in a CD20xCD3-sensitive human B lymphoma cell line. Among the most highly enriched sgRNAs were those targeting genes with predicted effects on cell-cell adhesion, including sialophorin (SPN). Increased expression of SPN impeded tumor cell clustering with T cells, thereby limiting CD3 bsAb-mediated tumor cell lysis. This inhibitory effect of SPN appeared to be dependent on sialylated core 2 O-glycosylation of the protein. While SPN is not endogenously expressed in the majority of B cell lymphomas, it is highly expressed in acute myeloid leukemia. CRISPR-mediated SPN knockout in AML cell lines facilitated T cell-tumor cell clustering and enhanced CD3 bsAb-mediated AML cell lysis. In sum, our data establish that the cell cross-linking mechanism of CD3 bsAb is susceptible to subversion by anti-adhesive molecules expressed on the tumor cell surface. Further evaluation of anti-adhesive pathways may provide novel biomarkers of clinical response and enable the development of effective combination regimens for this promising therapeutic class.

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Activation of the heat shock transcription factor by hypoxia in normal and tumor cell lines in vivo and in vitro

International Journal of Radiation Oncology*Biology*Physics ◽

10.1016/0360-3016(92)90667-7 ◽

1992 ◽

Vol 23 (4) ◽

pp. 891-897 ◽

Cited By ~ 19

Author(s):

Amato J. Giaccia ◽

Elizabeth A. Auger ◽

Albert Koong ◽

David J. Terris ◽

Andrew I. Minchinton ◽

...

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Tumor Cell ◽

Cell Lines ◽

Heat Shock Transcription Factor ◽

Tumor Cell Lines

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Tumor Cell-Dependent Differences in Stroma-Induced Changes to Bortezomib Sensitivity.

Blood ◽

10.1182/blood.v120.21.2468.2468 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2468-2468

Author(s):

Eugen Dhimolea ◽

Jana Jakubikova ◽

Richard W.J. Groen ◽

Jake E. Delmore ◽

Hannah M. Jacobs ◽

...

Keyword(s):

Tumor Cell ◽

Tumor Cells ◽

Cell Lines ◽

Stromal Cells ◽

Cell Response ◽

Research Funding ◽

Bioluminescence Imaging ◽

Bortezomib Treatment ◽

Rpmi 8226

Abstract Abstract 2468 In multiple myeloma (MM) and other hematologic malignancies, bone marrow stromal cells (BMSCs) confer resistance to diverse conventional or investigational therapeutics. During the last decade, data from many groups have concurred that the in vitro anti-MM activity of the proteasome inhibitor bortezomib is very similar in the presence and absence of BMSCs, including primary and immortalized BMSCs. These well-validated observations have supported the notion that novel, more effective, therapies for the treatment of MM should ideally be, similarly to bortezomib, capable of overcoming the protective effect of BMSCs. Interestingly, however, we have observed that primary CD138+ MM tumor cells isolated from patients with clinical refractoriness to bortezomib occasionally exhibit substantial in vitro response to clinically achievable concentrations of this drug. We therefore hypothesized that, under certain previously under-explored experimental settings, BMSCs may alter the threshold of MM cell response to bortezomib-induced apoptosis. To address this hypothesis in conditions that better simulate the clinical context, we conducted compartment-specific bioluminescence imaging (CS-BLI) assays to evaluate the effect of bortezomib on tumor cells co-cultured with BMSCs for different time periods prior to bortezomib administration. We observed that prolonged tumor-stromal co-culture (48–96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for several MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Prolonged co-culture of OPM1, RPMI-8226-Dox40, OCI-My5, KMS12BM and KMS18 cells prior to bortezomib treatment enhanced its activity. Importantly, extended co-culture of MM cell lines MM.1S and MM.1R with BMSCs prior to drug treatment induced significant attenuation of their response to bortezomib, as evidenced by 2–3 fold increase of IC50 values in several independent replicate experiments and a mean % area under the bortezomib dose response curve (AUC) of 5.82% vs 14.10% in the absence vs. presence of BMSCs, respectively (p=0.0079). Consistent with these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells mixed with Luc- BMSCs did not show statistically significant reduction in MM burden with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.1320), as quantified by bioluminescence imaging. In contrast, the same dose and schedule of bortezomib treatment significantly suppressed tumor burden, compared to vehicle-treated controls, of monotypic s.c. xenografts of Luc+ MM.1S cells in SCID mice (p=0.0022), as in prior experience. To evaluate the molecular mechanisms of cell non-autonomous decrease in MM cell response to bortezomib, we compared the transcriptional profiles of MM.1S cells in extended co-cultures with HS-5 BMSCs vs. MM.1S cells cultured in isolation. These studies identified a distinct transcriptional signature of stroma-induced transcripts, including several (e.g. PSMC3, ITGB7, FOS, ALDH1L2) for which transcript expression higher than the median levels for refractory MM patients correlated with shorter overall survival (p<0.02, log-rank tests) after treatment with bortezomib. These observations highlight the notion that tumor cell responses to a given agent in the presence of non-malignant stromal cells can exhibit substantial qualitative and quantitative variation, depending on the specific tumor cell type tested, as well as the particular stromal cell population and conditions of the co-culture. Our findings highlight the need to apply combinatorial high-throughput scalable platforms, such as CS-BLI, to evaluate the different permutations of interactions between tumor cells, non-malignant accessory cells of the microenvironment and administered therapeutics. This study also provides a comprehensive functional oncogenomic framework to identify prognostically relevant molecular mediators of stroma-induced resistance to therapy in MM. Disclosures: Groen: Genmab BV: Research Funding. McMilllin:Axios Biosciences: Equity Ownership. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor, Uncompensated Role as advisor Other.

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Lin28B/Let-7 Axis Regulates Multiple Myeloma Proliferation By Enhancing c-Myc and Ras Survival Pathways

Blood ◽

10.1182/blood.v122.21.273.273 ◽

2013 ◽

Vol 122 (21) ◽

pp. 273-273

Author(s):

Salomon Manier ◽

John T Powers ◽

Antonio Sacco ◽

Michaela R Reagan ◽

Michele Moschetta ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Loss Of Function ◽

Advisory Committees ◽

Expression Levels ◽

Gain And Loss

Abstract Background MicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. Lin28B is an RNA binding protein that regulates Let-7 miRNA maturation. Lin28B and Let-7 have been described to act as oncogenes or tumor suppressor genes, respectively, as demonstrated both in solid cancer and hematologic malignancies. However, the role of the Lin28B/Let-7 axis in Multiple Myeloma (MM) has not been studied. Method Lin28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Let-7 expression levels were assessed in CD138+ primary MM cells and bone marrow stromal cells (BMSCs) by using PCR, as well as in circulating exosomes using miRNA array (Nanostring® Technology). Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. The knockdown of Lin28B was performed on MM cell lines (U266, MM.1S, MOLP-8) by using a lentiviral Lin28B shRNA. Gain- and loss-of function studies for Let-7 were performed using Let-7 mimic and anti-Let-7 transfection in MM cell lines (MM1S, U266) and primary BMSCs. Cell proliferation has been evaluated by using thymidine assays. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot. Results Two independent GEP datasets (GSE16558; GSE2658) were analyzed for Lin28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high Lin28B levels correlated with a shorter overall survival (p = 0.0226). We next found that the Let-7 family members are significantly down-regulated in MM primary cells, particularly Let-7a and b (5 fold change, p < 0.05), as demonstrated by using qRT-PCR. Similarly, miRNA arrays showed a lower expression of Let-7-related miRNAs in circulating exosomes obtained from MM patients compared to healthy individuals. We further dissected the functional relevance of Lin28B in MM cells, by performing Lin28 knockdown (KD) in MM cell lines (U266, MOLP-8). This led to a significant decrease in MM cell proliferation associated with G1 phase cell cycle arrest. This was supported by up-regulation of Let-7 and down-regulation of c-Myc, Ras and Cyclin D1 in Lin28 KD MM cells. To further prove that Lin28B-dependent effects on MM cells are mediated by Let7, we next showed that let-7 gain- and loss-of-function studies regulate MM cell proliferation and Myc expression. Lin28B regulation in MM cells is dependent on Let-7, as demonstrated by an increase of both cell proliferation and c-Myc expression after anti-Let-7 transfection in the Lin28B KD cells. We therefore studied the regulation of Let-7 in MM cells through the interaction with BMSCs. Let-7 expression levels were significantly lower in BMSCs obtained from MM patients compared to healthy donors. Interestingly, the Let-7 expression level in MM cells was increased after co-culture with Let-7 over-expressing BMSCs, associated with a decrease of both cell proliferation and c-Myc expression. This suggests a potential transfer of Let-7 from BMSCs to MM cells. Conclusion This work describes a new signaling pathway involving Lin28B, Let-7, Myc and Ras in MM. Let-7 expression in MM cells is also regulated through the interaction of MM cells with BMSCs, leading to cell proliferation and Myc regulation in MM. Interference with this pathway might offer therapeutic perspectives. Disclosures: Leleu: CELGENE: Honoraria; JANSSEN: Honoraria. Daley:Johnson and Johnson: Consultancy, Membership on an entity’s Board of Directors or advisory committees; MPM Capital: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Verastem: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Epizyme: Consultancy, Membership on an entity’s Board of Directors or advisory committees; iPierian: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Solasia, KK: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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Evaluation Of The Novel, Orally Bioavailable Selective Inhibitor Of Nuclear Export (SINE) Verdinexor (KPT-335) In Spontaneous Canine Cancer: Results Of Phase I and Phase II Clinical Trials

Blood ◽

10.1182/blood.v122.21.5149.5149 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5149-5149

Author(s):

Cheryl London ◽

Luis feo Bernabe ◽

Sandra Barnard ◽

William Kisseberth ◽

Antonella Borgatti ◽

...

Keyword(s):

Biological Activity ◽

Tumor Cell ◽

Median Time ◽

Cell Lines ◽

Nuclear Export ◽

Research Funding ◽

Tumor Cell Lines ◽

Time To Progression ◽

Orally Bioavailable

Abstract SINE (Selective Inhibitors of Nuclear Export) block the activity of XPO1/CRM1, 1 of 7 nuclear export proteins in cells, forcing the nuclear retention of key tumor suppressor proteins (TSP), leading to selective apoptosis of tumor cells. The purpose of these studies was to evaluate the in vitro activity of SINE against canine tumor cell lines and investigate the biological activity of Verdinexor (KPT-335) in dogs with spontaneous cancers as proof of principle for human clinical studies with SINE. Several different canine tumor cell lines including those derived from Non-Hodgkin Lymphoma (NHL) exhibited growth inhibition and apoptosis in response to nanomolar concentrations of SINE; NHL cells were particularly sensitive with IC50 concentrations ranging from 2-42 nM. A Phase I clinical trial of Verdinexor was performed in dogs with cancer with an emphasis on NHL given in vitro activity demonstrated against the tumor cell lines. The maximum tolerated dose (MTD) was 1.75 mg/kg twice per week although biological activity was observed at 1 mg/kg. Clinical benefit including Partial Response (PR) and Stable Disease (SD) for at least 4 weeks was observed in 9/14 dogs with NHL with a median time to progression of 66 days (range 35-256). A dose expansion study was performed in 6 dogs with NHL given 1.5 mg/kg Verdinexor on a Monday/Wednesday/Friday (MWF) regimen; clinical benefit (PR + SD) was observed in 4/6 dogs with a median time to progression of 83 days (range 35-250+). Toxicities were primarily gastrointestinal in nature consisting of anorexia, weight loss, vomiting and diarrhea and were manageable with supportive care and dose modulation. A validated health related Quality of Life (QOL) form used to assess dogs during treatment demonstrated that the overall quality of life did not decrease in dogs in this study supporting the notion that clinical toxicities associated with Verdinexor are generally well tolerated. Based on these findings, a Phase IIb study was performed in 58 dogs with either newly diagnosed or relapsed NHL. Drug was administered initially at 1.5 mg/kg MWF, but this dosing regimen was changed to 1.25 mg/kg M/Th due to the high rate of anorexia and weight loss on the MWF regimen; dose escalation was permitted to 1.5 mg/kg on the M/Th regimen. The objective response rate was 34% (1 CR, 19 PR) with an additional 33 dogs experiencing SD for a minimum of 4 weeks, resulting in a of 91% disease control rate. While the median time to progression was approximately 5 weeks, 19 dogs (32%) remained on study drug for >8 weeks; several dogs continue to receive Verdinexor. Laboratory abnormalities were minimal and clinical toxicities were mild on the M/Th regimen. Together, these data provide robust evidence that the novel orally bioavailable XPO1 inhibitor Verdinexor exhibits single agent biological activity in a spontaneous large animal model of human NHL. Furthermore, Verdinexor was well tolerated even in the absence of supportive care, suggesting that SINE compounds could exhibit good long-term tolerability in people. Disclosures: London: Zoetis: Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Abbott: Honoraria. Modiano:Karyopharm: Research Funding. Saint-Martin:Karyopharm: Employment. McCauley:Karyopharm : Employment, Equity Ownership, Patents & Royalties. Shacham:Karyopharm : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment.

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