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The proteasome inhibitor bortezomib induces apoptosis in multiple myeloma cells and has transformed patient outcome. Using in vitro as well as in vivo immunodeficient and immunocompetent murine multiple myeloma models, we here show that bortezomib also triggers immunogenic cell death (ICD), characterized by exposure of calreticulin on dying multiple myeloma cells, phagocytosis of tumor cells by dendritic cells, and induction of multiple myeloma–specific immunity. We identify a bortezomib-triggered specific ICD gene signature associated with better outcome in two independent cohorts of patients with multiple myeloma. Importantly, bortezomib stimulates multiple myeloma cell immunogenicity via activation of the cGAS/STING pathway and production of type I IFNs, and STING agonists significantly potentiate bortezomib-induced ICD. Our study therefore delineates mechanisms whereby bortezomib exerts immunotherapeutic activity and provides the framework for clinical trials of STING agonists with bortezomib to induce potent tumor-specific immunity and improve patient outcome in multiple myeloma.Significance:Our study demonstrates that cGAS/STING-dependent immunostimulatory activity mediates bortezomib anti-myeloma activity in experimental models and associates with clinical response to bortezomib in patients with multiple myeloma. These findings provide the rationale for clinical evaluation of STING agonists to further potentiate anti–multiple myeloma immune response.See related commentary by Zitvogel and Kroemer, p. 405.This article is highlighted in the In This Issue feature, p. 403

Supp. table S1 shows immune genes regulated after treatment with bortezomib in vivo.

Supp. Fig. 1 shows that bortezomib induces ICD in vitro. Suppl. Fig.2 shows that bortezomib treatment of co-culture of dendritic and T cells does not induce T cell maturation. Suppl. Fig. 3 shows that bortezomib-induced MM cell death promotes proliferation of CD4+ and CD8+ naïve T cells. Suppl. Fig.4 shows that bortezomib treatment generates a robust immune response against MM cells. Suppl. Fig. 5 shows pathways enriched by bortezomib treatment in vivo. Suppl. Fig. 6 shows Gene Ontology analysis of ICD signature genes. Suppl. Fig. 7 shows that MM cells engage in a type-I IFN response after treatment with bortezomib. Suppl. Fig. 8 shows type-I IFN response after bortezomib in MM is STING dependent. Suppl. Fig. 9 shows STING agonists potentiate bortezomib-induced anti-tumor immunity.

PDF fie 148K, Description of additional genotypes of cells analyzed for gammaH2AX distribution and detailed instability phenotypes of heterozygous cells

PDF file 3766K, Analysis of gammaH2AX distribution, chromosomal instability phenotypes in heterozygous cells, and verification of genotypes in human cells

Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

Background: In normal cells, homologous recombination (HR) is tightly regulated and plays an important role in the maintenance of genomic integrity and stability through precise repair of DNA damage. RAD51 is a recombinase that mediates homologous base pairing and strand exchange during DNA repair by HR. Our previous data in multiple myeloma and esophageal adenocarcinoma (EAC) show that dysregulated HR mediates genomic instability. Purpose of this study was to investigate role of HR in genomic instability, chemoresistance and immune dysregulation in solid tumors including colon and breast cancers. Methods: The GEO dataset were used to investigate correlation of RAD51 expression with patient survival and expression of various immune markers in EAC, breast and colorectal cancers. RAD51 was inhibited in cancer cell lines using shRNAs and a small molecule inhibitor. HR activity was evaluated using a plasmid-based assay, DNA breaks assessed by evaluating expression of γ-H2AX (a marker of DNA breaks) and p-RPA32 (a marker of DNA end resection) using Western blotting. Genomic instability was monitored by investigating micronuclei (a marker of genomic instability). Impact of RAD51 inhibitor and/or a DNA-damaging agent was assessed on viability and apoptosis in EAC, breast and colon cancer cell lines in vitro and in a subcutaneous tumor model of EAC. Impact of RAD51 inhibitor on expression profile was monitored by RNA sequencing. Results: Elevated RAD51 expression correlated with poor survival of EAC, breast and colon cancer patients. RAD51 knockdown in cancer cell lines inhibited DNA end resection and strand exchange activity (key steps in the initiation of HR) as well as spontaneous DNA breaks, whereas its overexpression increased DNA breaks and genomic instability. Treatment of EAC, colon and breast cancer cell lines with a small molecule inhibitor of RAD51 inhibited DNA breaking agent-induced DNA breaks and genomic instability. RAD51 inhibitor potentiated cytotoxicity of DNA breaking agent in all cancer cell types tested in vitro as well as in a subcutaneous model of EAC. Evaluation by RNA sequencing demonstrated that DNA repair and cell cycle related pathways were induced by DNA breaking agent whereas their induction either prevented or reversed by RAD51 inhibitor. In addition, immune-related pathways such as PD-1 and Interferon Signaling were also induced by DNA breaking agent whereas their induction prevented by RAD51 inhibitor. Consistent with these observations, elevated RAD51 expression also correlated with that of genes involved in inflammation and other immune surveillance. Conclusions: Elevated expression of RAD51 and associated HR activity is involved in spontaneous and DNA damaging agent-induced DNA breaks and genomic instability thus contributing to chemoresistance, immune dysregulation and poor prognosis in cancer. Therefore, inhibitors of RAD51 have great potential as therapeutic agents for EAC, colon, breast and probably other solid tumors.

Genomic instability contributes to cancer progression and is at least partly due to dysregulated homologous recombination. Here, we show that an elevated level of ABL1 kinase overactivates the HR pathway and causes genomic instability in multiple myeloma (MM) cells. Inhibiting ABL1 with either shRNA or a pharmacological inhibitor (nilotinib) inhibits HR activity, reduces genomic instability, and slows MM cell growth. Moreover, inhibiting ABL1 rescues the HR dysregulation and genomic instability caused by melphalan, a chemotherapeutic agent used in MM treatment, and increases melphalan’s efficacy and cytotoxicity in vivo in a subcutaneous tumor model. In these tumors, nilotinib inhibits endogenous as well as melphalan-induced HR activity. These data demonstrate that inhibiting ABL1 using the clinically approved drug nilotinib reduces MM cell growth, promotes genome stability, increases the cytotoxicity of melphalan (and similar chemotherapeutic agents), and can potentially prevent or delay progression in MM patients.

Conventionally ventrally located spinal tumor is approached through anterior vertebrectomy which requires bony fixation and then immobilization for a couple of months. The alternative route to deal with such type of tumor is anterolaterally to avoid the surgical and nonsurgical complications. We are reporting a minimally invasive anterolateral approach for C2 neurofibroma in an 84-year-old patient. Postoperatively this patient did not require cervical brace and postoperative discomfort was minimal. It was observed that dumbbell-shaped cervical tumor with no intradural pathology and wide neural foramina could also be taken care through the anterolateral route which did not require bony fusion or immobilization, but the expertise of the surgeon is necessary for performing these types of minimally invasive procedure to achieve the best results.

Introduction: Posterior circulation aneurysm constitutes 15%–20% of all intracerebral aneurysms. With the advancement of endovascular techniques, the microsurgery for posterior circulation aneurysms has been pushed back a little. Even the International Subarachnoid Aneurysmal Trial gave support to the concepts of endovascular procedures, but microsurgical modality should not be discouraged. We present our institutional experience of microsurgical techniques on posterior circulation aneurysms. Materials and Methods: We performed a retrospective analysis of 37 patients of posterior circulation aneurysm from 2015 to 2019, referred to Bantane Hospital, Japan. We included all posterior circulation aneurysms such as basilar tip, basilar trunk, and vertebral artery-posterior inferior cerebellar artery (VA-PICA) aneurysms, admitted and treated with clipping or bypass and trapping. We assessed the outcome as measured by modified Rankin Score (mRS), complications, and mortality. Results: Out of 37 patients, 10 cases were a basilar tip, one case was the basilar trunk, and 26 cases were VA-PICA aneurysm. Intraoperatively, neuromonitoring, indocyanine green dye, dual-image videoangiography (DIVA), and neuro endoscope were used. Two patients of basilar tip aneurysm developed third cranial nerve paresis and six patients of VA-PICA aneurysm developed lower cranial nerve paresis which resolved spontaneously. All the patients were discharged with mRS of 0 or 1. No mortality was recorded in our study. Conclusion: Microsurgical clipping of posterior circulation aneurysm is safe in unruptured aneurysm with a very low risk of mortality and morbidity under experienced hands. All postoperative complications in our study were transient and resolved with time with no residual deficits. Preoperative simulation, intraoperative neuromonitoring, DIVA, and neuro endoscope help achieve complete obliteration of aneurysmal sac and avoid complications.

Middle cerebral aneurysms constitute almost one-third of all anterior circulation aneurysms. Most of the saccular aneurysms originate from the arterial branching sites, but origins other than at the branching site are extremely rare. In this article, we are describing a unique M1 segment middle cerebral artery aneurysm which is not related with any branching site. Our literature search suggests that atherosclerotic changes in the arterial wall and local hemodynamic forces play an important role in the development of these types of aneurysm. Surgical management is not so unique in this type of aneurysm, but due to atherosclerotic parent arterial wall and thin-walled aneurysm sac, a neurosurgeon should be more cautious.

Identification of modifier genes predisposing to breast cancer (BC) phenotype remains a significant challenge and varies with ethnicity. The genetic variability observed in DNA repair genes may modulate the cell's ability to repair the damaged DNA and hence, evaluation of genetic variants in crucial DNA damage repair genes is of clinical importance. We performed the present study to evaluate the role of ERCC2-Lys751Gln, hOGG1-Ser326Cys, and XRCC1-Arg399Gln gene polymorphisms on the risk of BC development and its molecular profile in Indian women. Three non-synonymous variants (rs13181, rs1052133, and rs25487) were genotyped in 464 BC patients and 450 healthy controls. Logistic regression was employed to evaluate the association of genotypes with BC risk. Also, in silico analysis was carried out to map the Arg399Gln variant on the BRCT1 domain of XRCC1 protein. XRCC1 Gln/Gln genotype frequency was significantly elevated in BC patients [odd ratio (OR) = 1.73; 95% confidence interval (CI) = 1.13-2.65]. No significant association was observed between hOGG1-Ser326Cys and ERCC2-Lys751Gln variants and BC risk. Subgroup analysis revealed that ERCC2-Lys751Gln and XRCC1-Arg399Gln variants contributed towards tumor progression. A positive interaction between the investigated SNPs and BC was revealed by MDR analysis. Arg399Gln variant resulted in a change in the surface charge of XRCC1 protein. The rs25487 variant of XRCC1 might be associated with an elevated risk of BC. Furthermore, we demonstrated that high order gene-gene interaction plays a significant role in BC etiology. Hence, understanding the impact of low penetrant gene polymorphisms might enable a better understanding of the genetic background of breast cancer.

Background: Diffusion tensor imaging (DTI) and diffusion tensor tractography (DTT) provide a noninvasive window to study the neural connectivity and reconstruct the tracts. Detection of white matter injury (WMI) by DTT is a recent application being used in stroke, diffuse axonal injury, and neurodegenerative disorders. Fiber tracking in patients with brain hemorrhage can detect loss of fibers and anatomical disruption of the tracts, which can be useful in the prognostication of patient outcome. Materials and Methods: DTI and fiber tracking was done in four patients admitted at Fujita Health University Banbuntane Hospital, Japan, with decreased consciousness following brain hemorrhage (3 patients with aneurysmal subarachnoid hemorrhage and one patient with bifrontal hemorrhage), and WMI was analyzed. We also reviewed the literature on tractography in patients with brain hemorrhage and its correlation with consciousness. Results: We found significant frontal WMI in the form of thinning and anatomical disruption in all four cases. The frontal white matter tracts form an important component of the limbic system and ascending reticular activating system and frontal WMI correlated with the poor conscious level and cognitive dysfunction. Structural damage to the fiber tracts demonstrated as thinning, reduction in the volume or absence on tractography with corresponding reduction in the mean fractional anisotropy values in the frontal white matter of the affected side. Conclusion: DTI can be useful as a critical tool for revealing the anatomical basis for the cognitive dysfunction and unconsciousness and can be possibly used to prognosticate patient recovery. Early detection of WMI by DTI can also help in tailored rehabilitation. The authors believe that DTT could have a crucial role in the future for detecting structural changes which lead to cognitive dysfunction and further studies are needed to arrive at a specific protocol for detecting WMI.

Genomic instability fuels genomic alterations that befit cancer cells with necessary adaptations to keep proliferating and overcome the impact of host anti-tumor immunity and cytotoxic therapy. Since DNA breaks are required for genomic rearrangements to take place, we hypothesized that dysregulated nuclease activity mediates genomic instability in cancer. Using an integrated genomics protocol, we identified a four gene deoxyribonuclease signature correlating with genomic instability in six human cancers which included adenocarcinomas of esophagus (EAC), lung, prostate, stomach, pancreas and triple negative breast cancer. Functional screens confirmed the role of these nucleases in genomic instability and growth of cancer cells. Apurinic/apyrimidinic nuclease 1 (APE1), identified as top nuclease in functional screen, was further investigated in five cell lines representing four solid tumors (EAC, lung, prostate and breast cancer). We demonstrate that chemical as well as transgenic suppression of APE1 impaired growth/colony formation and increased cytotoxicity of chemotherapeutic agent, whereas inhibited spontaneous as well as chemotherapy-induced DNA breaks, homologous recombination (HR) activity and genomic instability in all cancer cell types tested. Treatment with APE1 inhibitor also impaired tumor growth and significantly increased efficacy of a chemotherapeutic agent in a subcutaneous mouse model of EAC. Overexpression of APE1 in normal esophageal epithelial cells increased DNA breaks and HR activity, leading to massive mutational, copy number as well as karyotypic instability. Evaluation of by whole genome sequencing identified HR as the top mutational process activated by APE1. Normal cells overexpressing APE1 grew as tumors in mice and tumors removed from mice displayed additional karyotypic changes, providing evidence of genomic instability in vivo. Overall, our data demonstrate that elevated APE1 dysregulates HR activity, G2/M checkpoint and genome stability thus contributing to tumorigenesis and chemoresistance in cancer. Therefore, inhibitors of APE1 have potential to inhibit growth and increase cytotoxicity of chemotherapeutic agents while minimizing spontaneous as well as chemotherapy-induced genomic damage and instability in EAC and other solid tumors.

SUMMARYLong noncoding RNAs (lncRNA) can drive the tumorigenesis and be susceptible to therapeutic intervention. To define the landscape of therapeutically actionable lncRNA dependencies in multiple myeloma (MM), we coupled our extensive lncRNA transcriptomic profile with lncRNA targeted CRISPR interference viability screen and identified RNA Regulator of Lipogenesis (RROL) as a leading lncRNA dependency in MM. RROL shares its origin with the microRNA locus MIR17HG, however supports the proliferation and survival of MM cells in a microRNA- and DROSHA- independent manner. We found that RROL provides a chromatin scaffold for the functional interaction between c-MYC and WDR82 to promote the regulation of the lipogenic pathways via the transcriptional control of the rate-limiting enzyme ACC1 in MM cells. Inhibition of RROL with clinically applicable antisense molecules disrupts its transcriptional and functional activities causing potent anti-tumor effects both in vitro and in vivo in two pre-clinical animal models. This study establishes lncRNA RROL as a therapeutically actionable dependency with a unique mechanism of action in support of myeloma cell growth.

The frequency of triple-negative breast cancer (TNBC) incidence varies among different populations, suggesting the involvement of genetic component towards TNBC development. Previous studies have reported that BRCA1/2 germline mutations confer a lifetime risk of developing TNBC. However, there is hardly any information regarding the common pathogenic variants (PVs) in BRCA1/2 genes that contribute to TNBC in the Indian population. Hence, we screened for PVs in BRCA1/2 and their association with clinico-pathological features in TNBC patients. Among the 59 TNBC genomic DNA samples sequenced, 8 BRCA mutations were detected in 59 TNBC patients (13.6%). Almost 50% pre-menopausal TNBC patients had a BRCA mutation. Of the 8 BRCA mutations, we observed BRCA1 mutations in 6 TNBC patients, and BRCA2 mutations in 2 TNBC patients. Among the 6 BRCA1 mutations, three were c.68_69delAG (185delAG) mutation. Remarkably, all the TNBC patients with BRCA mutations exhibited higher-grade tumors (grade 2 or 3). However, among the BRCA mutation carriers, only one patient with a BRCA2 mutation (p.Glu1879Lys) developed metastasis in the observed cohort. Our data advocates that South Indian women with higher grade TNBC tumors and without hereditary breast and ovarian cancer should be considered for BRCA mutation screening, thereby enabling enhanced decision-making and preventive therapy.

Aim In normal cells, homologous recombination (HR) is strictly regulated and precise and plays an important role in preserving genomic integrity by accurately repairing DNA damage. RAD51 is the recombinase which mediates homologous base pairing and strand exchange during DNA repair by HR. We have previously reported that HR is spontaneously elevated (or dysregulated) in esophageal adenocarcinoma (EAC) and contributes to ongoing genomic changes and instability. The purpose of this study was to evaluate the impact of RAD51 inhibitor on genomic toxicity caused by etoposide, a chemotherapeutic agent. Methods EAC cell lines (FLO-1 and OE19) were cultured in the presence of RAD51 inhibitor and/or etoposide, and impact on cell viability, apoptosis and genomic integrity/stability investigated. Genomic integrity/stability was monitored by evaluating cells for γ-H2AX (a marker for DNA breaks), phosphorylated RPA32 (a marker of DNA end resection which is a distinct step in the initiation of HR) and micronuclei (a marker of genomic instability). Results Treatment with etoposide, a chemotherapeutic agent, was associated with marked genomic toxicity (as evident from increase in DNA breaks) and genomic instability in both EAC cell lines. Consistently, the treatment was also associated with apoptotic cell death. A small molecule inhibitor of RAD51 increased cytotoxicity while reducing genomic toxicity and instability caused by etoposide, in both EAC cell lines. Conclusion RAD51 inhibitors have potential to increase cytotoxicity while reducing harmful genomic impact of chemotherapy.

Esophageal adenocarcinoma (EAC) is associated with a marked genomic instability, which underlies disease progression and development of resistance to treatment. In this study, we used an integrated genomics approach to identify a genomic instability signature. Here we show that elevated expression of this signature correlates with poor survival in EAC as well as three other cancers. Knockout and overexpression screens establish the relevance of these genes to genomic instability. Indepth evaluation of three genes (TTK, TPX2 and RAD54B) confirms their role in genomic instability and tumor growth. Mutational signatures identified by whole genome sequencing and functional studies demonstrate that DNA damage and homologous recombination are common mechanisms of genomic instability induced by these genes. Our data suggest that the inhibitors of TTK and possibly other genes identified in this study have potential to inhibit/reduce growth and spontaneous as well as chemotherapy-induced genomic instability in EAC and possibly other cancers., Subodh Kumar et al. identify a gene signature correlated with genomic instability and poor survival in esophageal adenocarcinoma (EAC), using a combination of integrative genomic analysis of patient data and laboratory validation in cell line models and mice. They find that inhibitors of some of the identified proteins, including TTK, could be used to reduce genomic evolution as well as inhibit growth of EAC cells.

Introduction Long noncoding RNAs (lncRNAs) are profoundly dysregulated in multiple myeloma (MM), yet their tumor-promoting features remain to be defined. Here, we have coupled extensive transcriptomic profiling of patient samples (n=360) with a genome-wide CRISPR interference (CRISPRi) viability screen (4 cell lines) to generate a map of lncRNA "dependencies" in MM; moreover, we describe the molecular and functional role – as well as its therapeutic potential – of the screen top hit RNA Regulator of Lipogenesis (RROL), a nuclear-retained lncRNA generated by alternative splicing of MIR17HG. Methods Loss-of-function (LOF) studies using antisense oligonucleotides (ASO) confirmed the RROL dependency in a large panel of MM cell lines and CD138+ patient cells, in vitro and in vivo in animal models. This effect was not antagonized in DROSHA KO MM cells or by ectopic expression of MIR17HG-derived miR-17-92, indicating a microRNA-independent role of RROL. Results Transcriptomic analysis after RROL depletion indicated a significant impact on the de novo lipogenesis (DNL) gene networks. RROL occupancy at DNL gene loci was detected by chromatin isolation by RNA precipitation followed by qRT-PCR (ChIRP-qPCR) and confirmed by DUAL RNA FISH analysis. At functional level, we demonstrated that RROL depletion reduces DNL in MM cells using an unbiased lipidomic profiling as well as by measuring the incorporation of C14-radiolabeled glucose into lipids. Importantly, we also proved that exogenous palmitate, the downstream product of DNL pathway, can significantly rescue the growth inhibitory effect of RROL depletion in MM cells; thus implicating a role for DNL in the growth promoting effect of this lncRNA. RNA-Protein Pull Down (RPPD) and in vivo RNA yeast three-hybrid (Y3H) assays led to identify c-MYC (MYC) as a relevant protein interactor of RROL; a finding validated by RIP-qPCR. Coupling RNA FISH with immunofluorescence, we co-detected RROL and MYC at the ACC1 promoter. Importantly, neither RROL or MYC could occupy ACC1 promoter and exert regulatory control in the absence of the other factor. Moreover, using in vitro (Co-IP/MS) and in vivo (BioID) assays, we identified WDR82 as an RROL-dependent MYC partner implicated in the transcriptional control of ACC1 expression. Finally, to therapeutically antagonize RROL, we have screened >100 ASOs and provided the basis to develop a first-in-class RROL inhibitor. This inhibitor has shown a very strong anti-MM activity in vitro (IC50 Conclusion In conclusion, we here report a unique regulatory function of a novel lncRNA supporting MM cell growth via its control of the lipogenic metabolic axis. The ongoing development of RROL inhibitors may allow clinical application of this unique targeted therapy in MM.

Background We have previously reported the identification of FEN1 a driver of genomic instability in myeloma (MM) as well as esophageal adenocarcinoma (EAC) cells. We demonstrated that FEN1 is overexpressed in MM cell lines and in clinical datasets of MM and several other cancers including EAC, and FEN1-knockdown inhibited spontaneous DNA breaks, homologous recombination (HR) activity as well as genomic instability in MM cells. We now show that FEN1-overexpression in non-cancerous (normal fibroblasts and bone marrow/stroma HS5) cells increases DNA breaks and genomic instability, as assessed by micronucleus assay. Methods In order to further evaluate the impact on genomic instability, control and FEN1-overexpressing cells were cultured for three weeks and new genomic changes acquired in cultured relative to “day 0” cells (representing baseline genome), were identified using single nucleotide polymorphism (SNP) arrays. Results Overall, the acquisition of amplification and deletion events were increased by ~ 3-fold in FEN1-overexpressing relative to control cells. Evaluation by RNA sequencing in two different non-cancerous cell types showed that FEN1-overexpression was associated with upregulation of several interconnected pathways including DNA double strand break repair, cell cycle, mitotic G2 M phases, TP53, homologous DNA pairing and strand exchange. Top downregulated pathways included several metabolic pathways and an apoptosis pathway. These data demonstrate a significant role and the impact of FEN1 on DNA repair and genome maintenance, especially HR. We next identified FEN1-interacting proteins from two different MM cell lines (MM1S, RPMI8226) by mass spectrometry. Forty-one proteins interacted with FEN1 in both MM cell lines including two helicases (ASCC3, RUVBL2) and several proteins involved in DNA damage response and recruitment. To investigate the functional relevance of FEN1-ASCC3 interaction, FEN1 was overexpressed in non-cancerous (stromal HS5) cells and ASCC3 was suppressed in control as well as FEN1-overexpressed cells, and impact on different parameters of genome stability (HR activity, micronuclei) and growth (cell viability and DNA replication) monitored. FEN1-overexpression increased HR activity, whereas ASCC3-knockdown inhibited spontaneous as well as FEN1-induced HR activity. Consistent with these data, FEN1-overexpression also increased genomic instability, whereas ASCC3-knockdown inhibited spontaneous as well as FEN1-induced genomic instability as assessed by micronucleus assay. Importantly, FEN1-overexpression also increased DNA replication (as assessed by BrdU-labelling), whereas ASCC3-knockdown inhibited spontaneous as well as FEN1-induced DNA replication in these cells. Conclusion These data suggest that helicase activity of ASCC3 is coupled to endonuclease activity of FEN1 to cause genomic instability and cancer cell proliferation, and is currently being investigated in MM and other cancer models to develop translational application.

Until now, no study has reported the combined effect of genetic variants of HOTAIR and NME1 towards breast cancer (BC) pathogenesis. Hence, the aim of the present study is to determine the risk of breast cancer development with HOTAIR (rs920778 C T and rs1899663 G T) and NME1 (rs16949649 T C and rs2302254 C T) genetic polymorphisms in the Indian population for the first time.To investigate the genetic association of these four SNPs, we conducted a population-based case-control study involving 1011 subjects (502 histologically confirmed BC patients and 509 disease-free controls) using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.HOTAIR rs920778 TC genotype elevated the risk of BC (OR = 1.39, 95% CI = 1.06-1.83, p = 0.018) and individuals carrying the mutant allele (T) of rs1899663 had increased BC risk (OR = 1.23, 95% CI = 1.02-1.47, p = 0.026). The presence of the NME1 rs16949649 CC genotype increased the risk of BC (OR = 1.76, 95% CI = 1.15-2.71, p = 0.009). Moreover, the HOTAIR rs920778 variant (TC + CC) increased the risk of BC in pre-menopausal women (OR = 5.86; p 0.0001). Women carrying 2 or 3 mutant alleles for the investigated SNPs were observed to have an elevated risk of BC.The results of the present study highlight the presence of significant associations between NME1 rs16949649 and HOTAIR (rs920778 and rs1899663) polymorphisms and breast cancer development in Indian women.

Identification of genes driving genomic evolution can provide novel targets for cancer treatment and prevention. Here we show identification of a genomic instability gene signature, using an integrated genomics approach. Elevated expression of this signature correlated with poor survival in esophageal adenocarcinoma (EAC) as well as three other human cancers. Knockout and overexpression screens confirmed the relevance of this signature to genomic instability. Indepth evaluation of TTK (a kinase), TPX2 (spindle assembly factor) and RAD54B (recombination protein) further confirmed their role in genomic instability and tumor growth. Mutational signatures identified by whole genome sequencing and functional studies demonstrated that DNA damage and homologous recombination were common mechanisms of genomic instability induced by these genes. Consistently, a TTK inhibitor impaired EAC cell growth in vivo, and increased chemotherapy-induced cytotoxicity while inhibiting genomic instability in surviving cells. Thus inhibitors of TTK and other genes identified in this study have potential to inhibit/delay genomic evolution and tumor growth. Such inhibitors also have potential to increase chemotherapy-induced cytotoxicity while reducing its harmful genomic impact in EAC and possibly other cancers.

The histone modifications and associated changes in chromatin structure and function have emerged as important epigenetic mechanisms impacting gene expression and have significant translational relevance in cancers, including multiple myeloma (MM). Epigenetic intervention with histone deacetylases (HDACs) inhibitors is emerging as a promising therapeutic strategy in combination with current anti-myeloma agents. Although pan-HDAC inhibitors have been shown to be effective both in preclinical and clinical setting, they seem to be associated with toxicity. It is, therefore, extremely important to understand the biological and molecular roles of individual HDACs to then selectively target them to limit toxicities observed with pan-HDAC inhibitors. Based on our observation that elevated HDAC8 expression correlates with poor overall survival in MM patients in three different datasets including one publicly available dataset (GSE39754), we evaluated its functional role in MM. HDAC8, a member of class I HDAC isoenzymes, is responsible for the deacetylation of lysine residues on the N-terminal part of the core histones as well as non-histone proteins. We performed genetic modulation of HDAC8 by loss-of-function studies, using shRNA as well as siRNAs targeting HDAC8. Downregulation of HDAC8 in 3 different MM cell lines caused MM cell growth inhibition in a time-dependent manner which was associated with induction of cell apoptosis. Consistently, treatment with a selective and potent HDAC8 inhibitor (OJI-1) caused a significant inhibition of MM cell growth in a panel of 20 MM cell lines (IC50 = 80 nM) in a time- and dose-dependent manner, while having a minimal impact on six PBMC samples from healthy donors both in resting and activated state (IC50 = 150 nM). The mechanism of cell death was apoptosis as demonstrated by annexin-labeling. Importantly, both the HDAC8 knockdown and OJI-1 treatment inhibited DNA breaks as evidenced from γH2AX expression or a single cell gel electrophoresis method to visualize and quantitate DNA breaks. HDAC8 inhibition also caused inhibition of RAD51 foci and HR activity, as measured by strand-exchange assay. Interestingly, non-homologous end joining in MM cells was not impacted by these treatments. Consistent with these data, the overexpression of HDAC8 in MM as well as in normal cells increased DNA breaks and HR activity. Furthermore, the inhibition of HDAC8 (by knockdown and OJI-1) inhibited, whereas its overexpression increased genomic instability, as assessed by micronucleus assay, in surviving MM cells. We also demonstrate that HDAC8 interacts with RAD51 and impacts its acetylation. The treatment of MM cells with HDAC8 inhibitor (OJI-1) increased RAD51 acetylation. Next, we examined the in vivo efficacy of the HDAC8 conditional knockdown in a human xenograft mouse model, using H929 cells injected subcutaneously in SCID mice. HDAC8 knockdown not only caused a significant reduction in tumor growth but also increased survival (p=0.0016) compared to mice injected with control cells. Evaluation of tumors from these mice confirmed in vivo inhibition of DNA breaks and HR activity, and induction of apoptosis following HDAC8-knockdown. HDAC8 inhibitor OJI-1 also synergistically increased the cytotoxicity of existing MM drugs including dexamethasone, bortezomib and lenalidomide. In conclusion, our results demonstrate that elevated HDAC8 in MM cells is involved in inhibition of apoptosis but also contributes to increased DNA breaks and dysregulation of homologous recombination and genome stability. Therefore, HDAC8 is a novel target for therapeutic application in MM. Selective and potent HDAC8 inhibitor OJI-1 has shown a favorable therapeutic index with synergistic effect in combination with existing MM drugs. Disclosures Hajek: Pharma MAR: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Honoraria, Research Funding. Munshi: Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy; Abbvie: Consultancy; Adaptive Biotechnology: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy; Legend: Consultancy.

Integrated genomic analysis including whole genome and RNA sequencing in primary multiple myeloma (MM) cells have reported a universal loss of BCL7A gene in MM patients compared to normal plasma cells (PC). Our Genetic modulation in in-vitro and in-vivo MM models have also validated the loss of BCL7A as an oncogenic event responsible for acquisition of a more proliferative phenotype in MM cells. We performed a comparative mass spectrometric analysis confirming BCL7A as a member of the canonical m-SWI/SNF chromatin remodeling complex. We therefore performed the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) to assess genome-wide changes in DNA accessibility upon BCL7A gain or loss. We found that loss of BCL7A in wild-type KMS12BM and NCI-H929 cells resulted in enrichment of important transcription factor motifs in the transcriptionally active sites of chromatin. We identified 36 de novo accessible and 1079 de novo inaccessible regions across the genome after BCL7A shRNA KD (knock-down). These genomic regions with altered accessibility were associated with genes involved in protein binding (FDR < 0.001), GTPase activation (FDR = 0.016), and GTPase regulation (FDR = 0.030). Candidate transcription factors for the genomic regions with altered accessibility were identified by querying a database of human ChiP-seq experiments (ReMap 2020). IRF4 was identified to be enriched in regions of accessible chromatin upon BCL7A loss. IRF4 is an oncoprotein transcription factor and is a direct target of myc, generating a feedback loop in MM cells. IRF4 dependency is central to myeloma cell proliferation. Most importantly, we found that in addition to functions within the m-SWI/SNF complex, BCL7A forms a protein complex with IRF4. Altogether these data suggest a role for BCL7A in driving IRF4 oncogenic activities in MM. To understand how BCL7A-dependent changes may influence IRF4 activity, we performed CHiP assay using IRF4 antibody in BCL7A KO (knock-out) and AB (add-back) KMS12BM and NCI-H929 cells. We observed increased binding of IRF4 to the promoter of its target genes like PRDM1, CDK6, STAG2, PIM2, SQLE, Myc, CANX, IRF4, SCD, ELL2, CASP3 in BCL7A KO cells, while the binding of these target genes was significantly decreased in AB cells. While, CHiP assay using IRF4 antibody in control and ectopically expressed BCL7A in AMO1 and KMS11 cells showed significantly low binding of IRF4 to the promoter of most of its target genes in BCL7A overexpressed cells compared to control. Integrated transcriptomic analysis following BCL7A KD and overexpression revealed the existence of a set of genes transcriptionally regulated by IRF4 to be significantly upregulated following BCL7A depletion and downregulated following ectopic expression of BCL7A. To investigate whether these genes are involved in the phenotypic and functional effects observed in MM after BCL7A depletion, we performed LOF studies (si-RNA screen) in scrambled and BCL7A KD MM cells. Among others, we observed that MM cells are highly sensitive to the inhibition of EEF1B2, RPS3A, SOX2, DCC and NDUFA1 only in the context of BCL7A loss, implicating a role as critical effector molecules downstream of the IRF4-BCL7A transcriptional network. In conclusion, we observe that BCL7A binds to IRF4, functionally restricting its activity. The universally observed down regulation of BCL7A in MM provides the necessary molecular change to allow IRF4 to exert its required transcriptional activity to induce MM cell growth. Our results now provide the basis to understand the mechanism of development of IRF4 dependency in MM. Disclosures Anderson: Pfizer: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Janssen: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Takeda: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Pfizer: Consultancy.

Long noncoding RNA (lncRNAs) are key epigenetic factors that drive the origin and progression of human cancers via mechanisms that are largely unknown. We have previuosly reported the clinical significance of a lncRNA signature in multiple myeloma (MM) as independent risk predictor for clinical outcome; and recently identified a lncRNA RROL (RNA Regulator of Lipogenesis) with impact on MM cell proliferation. Here we describe a unique regulatory function for RROL in the control of gene networks involved in the de novo lipogenesis (DNL) pathway, ultimately impacting MM cell growth and survival. Based on growth and survival impact of RROL depletion, we performed integrated transcriptomic analysis of RNA-seq data after RROL depletion in MM cell lines and CD138+ patient MM cells, and identified a set of significantly modulated metabolic genes including the acetyl Co-A Carboxylase 1 (ACC1) gene, encoding a rate-limiting enzyme of the DNL pathway. This metabolic pathway converts nutrients into fatty acids serving for energy storage or biosynthesis of membranes and signaling molecules. Consistent with the transcriptional control of ACC1, we have observed that RROL inhibition in cell lines and primary MM cells significantly decreased the incorporation of C14-radiolabeled glucose into de novo synthesized lipids. Importantly, supplementation with exogenous palmitate, the main downstream product of DNL pathway, rescued the growth inhibitory effect of RROL depletion on MM cells, further confirming the importance of the DNL pathway in the oncogenic activity of RROL in MM. To understand the molecular mechanism through which RROL regulates ACC1 expression and its metabolic axis, we evaluated the RROL interactome in MM cells. RNA-Protein Pull Down (RPPD) and in vivo RNA yeast three-hybrid (Y3H) assays led to the identification of MYC as relevant direct partner of RROL. These results were further validated by qRT-PCR analysis of MYC-bound RNA obtained through RNA immunoprecipitation (RIP) assay. Using experimental model of conditional MYC KD (P493-6), we found that RROL exerts regulatory activity on ACC1 only in the presence of MYC. Mapping of MYC genomic occupancy by ChIP-seq and gene expression after MYC KD in MM cells revealed that ACC1 is a direct transcriptional target of MYC in cells expressing RROL. These data indicate that RROLand MYC cooperate in the transcriptional control of ACC1. Moreover, we found that RROL itself is transcriptionally regulated by MYC, suggesting the existence of a feed-forward regulatory loop in which MYC enhances the expression of RROL that, in turn, drives MYC transcriptional activity to ACC1. We hypothesized that RROL may shape the protein interacting network of MYC to confer specificity for ACC1 promoter. To this end, we performed mass spectrometry analysis of MYC interactome in three MM cell lines in the presence or in the absence of RROL and identified the transcriptional modulator WDR82 as RROL-dependent MYC partner. Interestingly, WDR82 directly interacts with RROL as assessed in the RPPD and RNA Y3H assays, and transcriptionally regulates ACC1 in RROL-dependent manner. These data indicate that RROL catalyzes the interaction of MYC with the transcriptional modulator WDR82 to form a transcriptional ternary complex regulating ACC1 expression. To therapeutically antagonize the RROL lipogenic signaling we have pre-clinically tested small molecule inhibitors of ACC1 (ACC1i). We have observed significant anti-MM activity of ACC1i in vitro in a large panel of MM cell lines and primary MM cells from patients; and in vivo in mouse models of human MM including the localized subcutaneous model and the disseminated model that establish a more aggressive systemic disease. Importantly, we have now developed clinically applicable ASOs and small molecule-like compounds to directly target RROL in MM cells. These studies are ongoing and will be presented. In conclusion, we here report a unique regulatory function of a novel lncRNA supporting MM cell growth via its control of the lipogenic metabolic axis. The availability of oral inhibitors of ACC1 as well as the ongoing development of RROL inhibitors may allow clinical application of this unique targeted therapy in MM. Disclosures Fulciniti: NIH: Research Funding. Chauhan:Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy.

Immune escape underlies progression of disease and resistance to therapy in MM, and dysfunction of both innate and adaptive immunity highlight the urgent need for scientifically-informed strategies to restore anti-MM immunity and improve patient outcome. Here we demonstrate that proteasome inhibitor bortezomib (BTZ) modulates the immunosuppressive bone marrow milieu by inducing immunogenic cell death (ICD) and a type-I interferon (IFN) response which contribute to its clinical efficacy. Moreover, we identified STING as a mediator of its anti-MM immune response, and provide the preclinical rationale for clinical trials of BTZ-STING agonist combination therapy. We first show that BTZ induces hallmarks of ICD in both human and murine MM cell lines, including exposure of endoplasmic reticulum protein calreticulin (CALR), that functions as an "eat me signal". Specifically, co-culture with BTZ-treated MM cells induced functional maturation of dendritic cells (DCs) and enhanced uptake of BTZ-treated MM cells, assessed by flow cytometry and confocal microscopy. Notably, these functional sequelae were abrogated when DCs were co-cultured with CALRKO MM cells; and add-back experiments by stable overexpression of CALR in KO clones confirmed the specific role of CALRin BTZ-induced immunogenicity. We next validated these findings in 2 in vivo syngeneic models. First, we observed that anti-MM activity of BTZ resulted in more potent murine 5TGM1 tumor cell shrinkage in immunocompetent than immunodeficient hosts, an effect mediated by ICD induction since it was abrogated in immunocompetent mice bearing CALRKO tumors. Second, in vitro BTZ-treated 5TGM1 cells were used as a vaccine to induce a protective anti-MM immune response: vaccination protected against tumor growth upon rechallenge with 5TGM1 cells; conversely, palpable tumors were detected in non-vaccinated mice by 1 week. Moreover, vaccination with BTZ-treated CALRKO 5TGM1 cells abrogated the protective effect of BTZ since only 50% mice were tumor free by 30 days. Consistently, ELISPOT assay on mice splenocytes confirmed that vaccination with BTZ-treated 5TGM1 cells triggered a MM-specific T cell response. Next, we performed RNAseq analysis of BTZ-treated vs untreated tumors from both CALRWT and CALRKO cells growing in immunocompetent mice; and then carried out an integrative analysis of RNAseq data from clinically-annotated MM patients (n=327) uniformly treated with BTZ-based regimens (IFM/DFCI 2009). We identified a specific ICD signature induced by BTZ only in CALRWT tumors in mice; and importantly, we found that increased expression of the human orthologs of this ICD signature was strongly and positively correlated with clinical outcome (overall survival (OS), p value=0.01). Moreover, the predictive value of this signature was confirmed in an independent dataset of BTZ-treated patients (GSE9782) (OS p value=0.024). Gene ontology analysis of the ICD signature genes revealed enrichment in inflammatory response pathways, including IFN stimulated genes (ISGs). Using RNAseq analysis and qRT-PCR validation, we showed that MM cells demonstrate a type-I IFN response after BTZ treatment which contributes to its in vivo efficacy, since neutralization of type-I IFNs signaling in both MM and host cells with a type-I IFNs receptor 1 (IFNAR) blocking antibody significantly reduced BTZ anti-MM activity. Finally, we showed that BTZ increases genomic instability/micronuclei formation in MM cells and activates innate cGAS/STING immune pathway, thereby stimulating a type-I IFN response. Conversely, STINGKO in MM cells abrogated this effect and decreased BTZ-triggered anti-MM T cell response. Moreover, expression of the ISGs included in ICD signature was positively correlated with STING expression in MM patients. Importantly, pharmacological activation of STING with STING agonist induced potent anti-MM activity in vivo; and combination of STING agonist with BTZ further potentiated this in vivo anti-MM response, with increased T cell infiltration into retrieved tumors, evidenced by IHC analysis. In conclusion, our studies delineate a novel mechanism whereby BTZ triggers anti-MM immune responses, and show that STING agonists can enhance this response. These findings provide the framework for clinical evaluation of STING agonists in combination with BTZ to induce potent anti-MM immune responses and thereby improve patient outcome. Disclosures Fulciniti: NIH: Research Funding. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Chauhan:Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Munshi:Karyopharm: Consultancy; Takeda: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Anderson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees.

Multiple myeloma (MM) is molecularly heterogenous disease with significant genomic instability. It carries number of mutations at diagnosis (median > 7000) and acquires additional changes overtime. With this background, we have evaluated the molecular intermediates of genomic instability in MM. Based on our large transcriptomic data we have identified apurinic/apyrimidinic deoxyribonuclease (APEX) as an important target whose elevated activity contributes to dysregulation of homologous recombination (HR) and genome stability in MM. Our investigation also demonstrates that transgenic overexpression of APEX nucleases induces genomic instability, leading to oncogenic transformation and tumorigenesis in a murine and Zebrafish models. Importantly, we have now observed that both transgenic as well as chemical inhibition of APEX1, reduces DNA breaks, HR activity and genomic instability as measured by micronucleus assay, and induces G2/M arrest in myeloma as well as esophageal cancer cells. To identify novel and effective inhibitors of APEX1, we optimized a high throughput APEX1 activity assay and screened a custom library of 100,000 small molecules. We identified API-93 as an effective APEX inhibitor in both the primary and secondary screens, and have now investigated it, alone as well as in combination, with existing myeloma drugs, for impact on different parameters of growth and genome maintenance. Although API-93 had minimal single agent cytotoxic effect on MM cells, it synergistically increased cytotoxicity of chemotherapeutic agent cyclophosphamide in both MM cell lines (MM1S and RPMI) tested, and also increased the efficacy of melphalan in several MM cell lines (RPMI, MM1R, KK1 and LR5) tested. A strong synergistic effect of API-93 was also observed in combination with lenalidomide in 5 MM cell lines tested (RPMI, MM1S, MM1R, KK1, H929; combination indexes < 1) as well as velcade in MM cells. For evaluation of impact on genomic changes, myeloma cells were treated with API-93, live cells purified and evaluated for impact on DNA breaks (by measuring levels of γ-H2AX), DNA end resection (a decisive step in the initiation of HR, by monitoring levels of p-RPA32), and genome stability by investigating micronuclei (the marker of genomic instability). Treatment with API-93 reduced spontaneous as well as melphalan-induced DNA breaks, DNA end resection as well as genomic instability (as assessed by significant reduction in micronuclei) in MM cells, in a dose-dependent manner. To further confirm the impact on genome stability, the MM cells were cultured in the presence or absence of API-93, and the acquisition of new copy number changes over 3 weeks were measured using SNP arrays, in the cultured relative to Day 0 cells (representing baseline genome). Relative to control cells, the cells treated with API-93 resulted in dose-dependent decrease in the acquisition of new copy number events, from 50% to > 90%. In conclusion, these data demonstrate APEX gene as being associated with MM cell survival and with genome instability. The novel APEX1 inhibitor, identified in a functional screen, provides an important tool to augment cytotoxicity of the current therapeutics while significantly decreasing genomic evolution in MM. Disclosures Munshi: Adaptive: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Legend: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy.

Long non-coding RNAs (lncRNAs) are an important novel class of non-coding RNAs having lengths of 200 nucleotides and low expression. The HOX Transcript Antisense Intergenic RNA (HOTAIR) is one of the most extensively studied lncRNAs found dysregulated in human cancer. Although a growing body of evidence suggests a role fo HOTAIR in pathogenesis, disease progression, drug resistance and reduced survival, its mechanism of action remains largely unclear. Recent studies have identified that HOTAIR facilitates protein-protein interaction thereby affecting diverse pathways in cancer such as epigenetic reprogramming, protein stability and signal transduction. HOTAIR has been shown to promote tumor progression by regulating microRNA expression and function. Moreover, several HOTAIR gene variants have recently been identified and found to increase cancer susceptibility. Here we review recent data on the critical role of HOTAIR in human malignancy and its potential mechanism of action. A more comprehensive understanding of this unique lncRNA is critical to elucidating the pro-oncogenic function of HOTAIR its potential application in diagnosis, prognosis and treatment.

Multiple myeloma (MM) is a biologically heterogeneous plasma cell malignancy, however, the mechanisms underlying this complexity are incompletely understood. By deep (70x) whole-genome sequencing (WGS) of 312 primary MM patients, we observed mutations in about 62% of patients in the 5' untranslated region and intron 1 of the 12q24.3 region which cytogenetically marks the BCL7A gene. Integration of WGS with RNA-seq data suggested a widespread loss of BCL7A expression in MM cells as compared to normal plasma cells (PC). To understand the effect of noncoding mutations in BCL7A promoter we performed in-vitro luciferase reporter assay in MM cell lines (H929, MM1S and KK1) devoid of BCL7A mutation in 5'UTR and found that the normalized luciferase reporter activity indicated that the mutation in the promoter significantly reduced BCL7A promoter activity. Next, we recapitulated the loss of BCL7A observed in MM patients, in a panel of MM cell lines using shRNA based genetic interference and observed the appearance of a more proliferative phenotype. The effects of BCL7A loss in MM cells were further confirmed using CRISPR-Cas9 system. BCL7A-KO (knock-out) cells had higher proliferative rate compared to wild type (WT) cells and lentiviral add back of BCL7A plasmid reversed this effect. This phenomenon was validated in a in vivo model, where we found that mice bearing BCL7A KO MM cells developed tumors faster than mice injected with control or add-back cells, suggesting that BCL7A loss increases tumorigenesis in vivo. On the other hand, ectopic expression of BCL7A significantly reduced cell viability and colony formation over time, inducing apoptotic cell death and impacting genes involved in chromatin and chromosome organization, DNA repair and cell cycle. Interestingly, BCL7A is an important member of the m-SWI/SNF chromatin remodeling complex. Using comparative mass spectrometry analysis, we observed that BCL7A expression is essential for the formation of a functional canonical m-SWI/SNF complex in MM cells, controlling the incorporation of active sub-units into the complex. These data suggest that loss-of-function mutations in the BCL7A region may disrupt the formation of SWI/SNF complex activating a transcriptional program that leads to MM cell growth and viability. We therefore evaluated the transcriptomic changes in MM cells upon BCL7A perturbation in a panel of MM cell lines by whole genome RNA-seq. Integrated analysis of modulated genes identified a set of 23 genes significantly upregulated in the presence of BCL7A depletion and downregulated in presence of BCL7A ectopic expression. To investigate whether these genes are involved in the phenotypic and functional effects observed in MM after BCL7A depletion, we performed LOF (loss-of-function) studies (si-RNA screen) using these genes in scrambled and BCL7A KD (knock-down) MM cells. Among others, we observed that MM cells are highly sensitive to the inhibition of RPS3A (V-Fos transformation effector protein) only in the context of BCL7A loss. Our RNA-seq data revealed that RPS3A is highly expressed in primary MM cells, and its expression significantly correlates with low expression of BCL7A in MM patients. Importantly, we observed that RPS3A binds to the core m-SWI/SNF complex proteins; SMARCC2 and SMARCB1 in the absence of BCL7A. RPS3A gene encodes a ribosomal protein that is a component of the 40S subunit and its overexpression transforms NIH3T3 mouse fibroblasts and induces tumor formation in nude mice and also used as an expression marker in squamous cell carcinoma. In conclusion, we here report biological consequences of a frequent non-coding mutation of BCL7A in MM and loss-of-function mutations in the BCL7A region may disrupt the formation of SWI/SNF complex activating a transcriptional process that leads to MM cell growth and viability. Altogether this study shed lights on a new pathogenic mechanism that may drive MM growth with potential translational implication, that may be applicable to other hematological diseases. Disclosures Anderson: Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Fulciniti:NIH: Research Funding. Munshi:BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

Dysregulated homologous recombination (HR) contributes to the acquisition of genomic changes and development of drug resistance over time in multiple myeloma (MM). We have now further investigated the molecular intermediates that may play an important role in HR process. We observe that ABL kinase, which regulates RAD51 through its phosphorylation, contributes to increased HR activity and genomic instability in myeloma. Moreover, Cellular response after DSBs involves nuclear re-localization of ABL1 upon DNA damage. A cellular localization of ABL1 has been confirmed in MM. Consistently, we observe that ABL kinase inhibitor reduces HR activity and genomic instability (as assessed by micronucleus assay) in MM cells. Based on our observation that melphalan increases genomic instability (as assessed by micronuclei assay and by whole genome sequencing), we sought to investigate impact of melpahalan on HR and role of ABL1 kinase in this process. We show that treatment with melphalan leads to increase in RAD51 expression and HR activity in MM cells in a dose-dependent manner. Evaluation by RNA sequencing showed that treatment of MM1S cells with melphalan is associated with upregulation of p53 signaling (containing multiple genes involved in DNA damage, detection of damaged sites, recombination/repair and genomic instability) as the topmost pathway. These findings suggest that melphalan-induced DNA damage leads to a concerted overexpression of genes involved in DNA damage response, recombination, genomic instability and chemoresistance. With the role of ABL1-kinase following DSB, we investigated and report that treatment with melphalan induces micronuclei formation, whereas nilotinib significantly reduces both the basal and melphlalan-induced micronuclei in all MM cell lines tested. We have now confirmed these observations by evaluating copy number alterations using single nucleotide polymorphism (SNP) arrays. To evaluate the impact of nilotinib, melphalan and their combination on genomic instability, we cultured MM cells in the presence of nilotinib (2.5 µM), melphalan (1 µM) and combination of both drugs for three weeks and investigated the acquisition of new copy number events, relative to "day 0" cells (serving as baseline genome), using SNP arrays. Treatment with melphalan led to massive increase in the acquisition of amplification and deletion events, whereas nilotinib not only reduced the acquisition of copy number events under spontaneous condition but also almost completely reversed/prevented those induced by melphlalan. Importantly, the treatment with nilotinib could also significantly sensitized MM cell lines and bone marrow plasma cells from relapsed MM patients to melphalan treatment. These data confirm that ABL1 inhibition reduces spontaneous and melphalan-induced genomic instability in MM cells. Since components of cell cycle play critical role in the maintenance of genome stability and growth, we investigated the impact of ABL1-inhibitor nilotinib, alone and in combination with melphalan, on cell cycle. Treatment of MM1S cells with melphalan for 48hrs led to 2.5-fold increase in the accumulation of cells in S-phase, suggesting an increase in replication stress by melphalan. Nilotinib reduced both the spontaneous and melphalan-induced fraction of S-phase cells by 27% (±2%) and 30% (±1%), respectively. Combined treatment with nilotinib and melphalan also increased sub G1 fraction of cells by 2.3-fold compared to those treated with melphalan alone, suggesting increased apoptosis in these cells. These data are consistent with our observation that treatment of MM cells with melphalan increase the phosphorylation of RPA32, a marker of replication stress while nilotinib reduces both the endogenous and melphalan-induced phosphorylated RPA32 level, suggesting that nilotinib might also be helpful in combating replication stress-mediated genomic instability. Taken together, these data demonstrate the critical role of ABL1-kinase in both spontaneous and drug (melphalan)-induced genomic instability and its inhibition could reduce/delay genomic evolution while enhancing cytotoxicity in multiple myeloma. Disclosures Fulciniti: NIH: Research Funding. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy.

Multiple myeloma (MM) is a genomically heterogenous malignancy characterized by a number of copy number alterations (CNA). Moreover, there is a clear evolution of genomic changes that may affect prognosis. To identify the drivers of this inherent genomic instability, we applied an integrated genomics approach utilizing genomic data for copy number changes and transcriptomic profile. We first identified genes whose expression correlated with total copy number events in a patient dataset (gse26863, n=246). We then applied those genes to identify the genes whose elevated expression correlated with both overall and event free survival in two different datasets (IFM70, n=170; gse2408, n=559). Elevated expression of this 30 gene signature also correlated with poor overall as well as event free survival in a third myeloma dataset (MMRF; P In summary, we demonstrate that elevated ATAD2 contributes to dysregulation of DNA repair and genome stability and is required for MM cell survival, indicating that it is a promising target to inhibit growth and reduce genomic evolution in myeloma. Disclosures Munshi: BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

A web of interconnected and flexible metabolic pathways helps in maintaining cellular homeostasis. These flexible metabolic pathways are further deregulated to ensure cell survival during clinical conditions such as diabetes and cancer. In fact, complex metabolic programming is a hallmark of both diabetes and cancer. Normally, glucose absorbed by cells is catabolized through glycolysis to form pyruvate. Pyruvate fuels the citric acid cycle in the mitochondria of aerobic cells. Diabetic condition is presented with a battery of glycolytic abnormalities. Glucose-6-phosphate derived from glucose is converted back to glucose and pumped out of the cell. Similarly, pyruvate is also converted back to glucose. Import of glucose via GLUT4 is also impaired during diabetes. In spite of decelerated glycolysis, gluconeogenesis also occurs in diabetes. In diabetic settings, cells adapt to using acetyl-CoA derived from fatty acids to fuel citric acid cycle. Cancer cells also present with several metabolic abnormalities where glucose-6-phosphate powers pentose phosphate pathway and contribute ribose required for the rapid proliferation of transformed cells. Cancer cells convert pyruvate to lactate, which is transported out of cells. Glutamine provides the majority of anaplerotic carbon for the citric acid cycle, and acetyl-CoA derived from citrate contributes to the synthesis of fatty acids. Both 3-phosphoglycerate and pyruvate are spared for the synthesis of amino acids. In the case of diabetes, cells uptake very less glucose compared with normal cells, whereas in case of cancer, cells consume excess glucose due to aerobic glycolysis. In this chapter, we will discuss the major components of the metabolic deregulations in diabetes and cancer.

We have previously reported that homologous recombination (HR) is dysregulated in multiple myeloma (MM) and contributes to genomic instability and development of drug resistance. We now demonstrate that base excision repair (BER) associated apurinic/apyrimidinic (AP) nucleases (APEX1 and APEX2) contribute to regulation of HR in MM cells. Transgenic as well as chemical inhibition of APEX1 and/or APEX2 inhibits HR activity in MM cells, whereas the overexpression of either nuclease in normal human cells, increases HR activity. Regulation of HR by AP nucleases could be attributed, at least in part, to their ability to regulate recombinase (RAD51) expression. We also show that both nucleases interact with major HR regulators and that APEX1 is involved in P73-mediated regulation of RAD51 expression in MM cells. Consistent with the role in HR, we also show that AP-knockdown or treatment with inhibitor of AP nuclease activity increases sensitivity of MM cells to melphalan and PARP inhibitor. Importantly, although inhibition of AP nuclease activity increases cytotoxicity, it reduces genomic instability caused by melphalan. In summary, we show that APEX1 and APEX2, major BER proteins, also contribute to regulation of HR in MM. These data provide basis for potential use of AP nuclease inhibitors in combination with chemotherapeutics such as melphalan for synergistic cytotoxicity in MM.