Venetoclax resistance leads to broad resistance to standard-of-care anti-MM agents, but not to immunotherapies.

Venetoclax is the first example of personalized medicine for multiple myeloma (MM), with meaningful clinical activity as a monotherapy and in combination in myeloma patients harboring the t(11:14) translocation. However, despite the high response rates and prolonged PFS, a significant proportion of patients eventually relapse. Here, we aimed to study adaptive molecular responses after the acquisition of venetoclax resistance in sensitive t(11:14) MM cell models. We therefore generated single-cell venetoclax-resistant t(11:14) MM cell lines and investigated the mechanisms contributing to resistance as well as the cells' sensitivity to other treatments. Our data suggests that acquired resistance to venetoclax is characterized by reduced mitochondrial priming and changes in BCL-2 family proteins' expression in MM cells, conferring broad resistance to standard-of-care anti-myeloma drugs. However, our results show that the resistant cells are still sensitive to immunotherapeutic treatments, highlighting the need to consider appropriate sequencing of these treatments following venetoclax-based regimens.

ABL1 kinase plays an important role in spontaneous and chemotherapy-induced genomic instability in multiple myeloma.

ABSTRACT: Genomic instability contributes to cancer progression and is at least partly due to dysregulated homologous recombination (HR). 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 short hairpin RNA or a pharmacological inhibitor (nilotinib) inhibits HR activity, reduces genomic instability, and slows MM cell growth. Moreover, inhibiting ABL1 reduces the HR activity 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, reduces genomic instability in live cell fraction, increases the cytotoxicity of melphalan (and similar chemotherapeutic agents), and can potentially prevent or delay progression in patients with MM.

Elevated APE1 Dysregulates Homologous Recombination and Cell Cycle Driving Genomic Evolution, Tumorigenesis, and Chemoresistance in Esophageal Adenocarcinoma.

BACKGROUND & AIMS: The purpose of this study was to identify drivers of genomic evolution in esophageal adenocarcinoma (EAC) and other solid tumors. METHODS: An integrated genomics strategy was used to identify deoxyribonucleases correlating with genomic instability (as assessed from total copy number events in each patient) in 6 cancers. Apurinic/apyrimidinic nuclease 1 (APE1), identified as the top gene in functional screens, was either suppressed in cancer cell lines or overexpressed in normal esophageal cells and the impact on genome stability and growth was monitored in vitro and in vivo. The impact on DNA and chromosomal instability was monitored using multiple approaches, including investigation of micronuclei, acquisition of single nucleotide polymorphisms, whole genome sequencing, and/or multicolor fluorescence in situ hybridization. RESULTS: Expression of 4 deoxyribonucleases correlated with genomic instability in 6 human cancers. Functional screens of these genes identified APE1 as the top candidate for further evaluation. APE1 suppression in EAC, breast, lung, and prostate cancer cell lines caused cell cycle arrest; impaired growth and increased cytotoxicity of cisplatin in all cell lines and types and in a mouse model of EAC; and inhibition of homologous recombination and spontaneous and chemotherapy-induced genomic instability. APE1 overexpression in normal cells caused a massive chromosomal instability, leading to their oncogenic transformation. Evaluation of these cells by means of whole genome sequencing demonstrated the acquisition of changes throughout the genome and identified homologous recombination as the top mutational process. CONCLUSIONS: Elevated APE1 dysregulates homologous recombination and cell cycle, contributing to genomic instability, tumorigenesis, and chemoresistance, and its inhibitors have the potential to target these processes in EAC and possibly other cancers.

High-dose melphalan treatment significantly increases mutational burden at relapse in multiple myeloma.

High-dose melphalan (HDM) improves progression-free survival in multiple myeloma (MM), yet melphalan is a DNA-damaging alkylating agent; therefore, we assessed its mutational effect on surviving myeloma cells by analyzing paired MM samples collected at diagnosis and relapse in the IFM 2009 study. We performed deep whole-genome sequencing on samples from 68 patients, 43 of whom were treated with RVD (lenalidomide, bortezomib, and dexamethasone) and 25 with RVD + HDM. Although the number of mutations was similar at diagnosis in both groups (7137 vs 7230; P = .67), the HDM group had significantly more mutations at relapse (9242 vs 13 383, P = .005). No change in the frequency of copy number alterations or structural variants was observed. The newly acquired mutations were typically associated with DNA damage and double-stranded breaks and were predominantly on the transcribed strand. A machine learning model, using this unique pattern, predicted patients who would receive HDM with high sensitivity, specificity, and positive prediction value. Clonal evolution analysis showed that all patients treated with HDM had clonal selection, whereas a static progression was observed with RVD. A significantly higher percentage of mutations were subclonal in the HDM cohort. Intriguingly, patients treated with HDM who achieved complete remission (CR) had significantly more mutations at relapse yet had similar survival rates as those treated with RVD who achieved CR. This similarity could have been due to HDM relapse samples having significantly more neoantigens. Overall, our study identifies increased genomic changes associated with HDM and provides rationale to further understand clonal complexity.

A MIR17HG-derived long noncoding RNA provides an essential chromatin scaffold for protein interaction and myeloma growth.

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.

RAD51 Is Implicated in DNA Damage, Chemoresistance and Immune Dysregulation in Solid Tumors.

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.

A standalone approach to utilize telomere length measurement as a surveillance tool in oral leukoplakia.

Oral squamous cell carcinoma (OSCC) is often preceded by a white patch on a surface of the mouth, called oral leukoplakia (OL). As accelerated telomere length (TL) shortening in dividing epithelial cells may lead to oncogenic transformation, telomere length measurement could serve as a predictive biomarker in OL. However, due to high variability and lack of a universal reference, there has been a limited translational application. Here, we describe an approach of evaluating TL using paired peripheral blood mononuclear cells (PBMC) as an internal reference and demonstrate its translational relevance. Oral brush biopsy and paired venous blood were collected from 50 male OL patients and 44 male healthy controls (HC). Relative TL was measured by quantitative PCR. TL of each OL or healthy sample was normalized to the paired PBMC sample (TL ratio). In OL patients, the mean TL ratio was significantly smaller not only in the patch but also in distal normal oral tissue, relative to healthy controls without a high-risk oral habit. Dysplasia was frequently associated with a subgroup that showed a normal TL ratio at the patch but significantly smaller TL ratio at a paired normal distal site. Our data suggest that evaluation of TL attrition using a paired PBMC sample eliminates the requirement of external reference DNA, makes data universally comparable and provides a useful marker to define high-risk OL groups for follow-up programs. Larger studies will further validate the approach and its broader application in other premalignant conditions.

Dysregulated APOBEC3G causes DNA damage and promotes genomic instability in multiple myeloma.

Multiple myeloma (MM) is a heterogeneous disease characterized by significant genomic instability. Recently, a causal role for the AID/APOBEC deaminases in inducing somatic mutations in myeloma has been reported. We have identified APOBEC/AID as a prominent mutational signature at diagnosis with further increase at relapse in MM. In this study, we identified upregulation of several members of APOBEC3 family (A3A, A3B, A3C, and A3G) with A3G, as one of the most expressed APOBECs. We investigated the role of APOBEC3G in MM and observed that A3G expression and APOBEC deaminase activity is elevated in myeloma cell lines and patient samples. Loss-of and gain-of function studies demonstrated that APOBEC3G significantly contributes to increase in DNA damage (abasic sites and DNA breaks) in MM cells. Evaluation of the impact on genome stability, using SNP arrays and whole genome sequencing, indicated that elevated APOBEC3G contributes to ongoing acquisition of both the copy number and mutational changes in MM cells over time. Elevated APOBEC3G also contributed to increased homologous recombination activity, a mechanism that can utilize increased DNA breaks to mediate genomic rearrangements in cancer cells. These data identify APOBEC3G as a novel gene impacting genomic evolution and underlying mechanisms in MM.

Integrated genomics and comprehensive validation reveal drivers of genomic evolution in esophageal adenocarcinoma.

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.

RAD51 Inhibitor Reverses Etoposide-Induced Genomic Toxicity and Instability in Esophageal Adenocarcinoma Cells.

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.

MUS81 Participates in the Progression of Serous Ovarian Cancer Associated With Dysfunctional DNA Repair System.

Objective: Methyl methanesulfonate ultraviolet sensitive gene clone 81 (MUS81) is a structure-specific endonuclease that plays a pivotal role in the DNA repair system of cancer cells. In this study, we aim to elucidate the potential association between the dysfunction of MUS81 and the progression of Serous Ovarian Cancer (SOC). Methods: To investigate the association between MUS81 and prognosis of SOC, immunohistochemistry technology and qPCR were used to analyze the level of MUS81 expression, and transcriptional profile analysis and protein interaction screening chip were used to explore the MUS81 related signal pathways. Random amplified polymorphic DNA (RAPD) analysis, immunofluorescence and comet assays were further performed to evaluate genomic instability and DNA damage status of transduced SOC cells. Experiments both in vitro and in vivo were conducted to verify the impact of MUS81 silencing on chemotherapeutic drug sensitivity of SOC. Results: The overexpression of MUS81 in SOC tissues was related to poor clinical outcomes. The transcriptional chip data showed that MUS81 was involved in multiple pathways associated with DNA repair. Deficiency of MUS81 intensified the genome instability of SOC cells, promoted the emergence of DSBs and restrained the formation of RAD51 foci in SOC cells with exposure to UV. Furthermore, downregulation of MUS81 enhanced the sensitivity to Camptothecin and Olaparib in SOC cell lines and xenograft model. Conclusions: MUS81 is involved in the progression of SOC and inhibition of MUS81 could augment the susceptibility to chemotherapeutic agents. MUS81 might represent a novel molecular target for SOC chemotherapy.

Dual PAK4-NAMPT Inhibition Impacts Growth and Survival, and Increases Sensitivity to DNA-Damaging Agents in Waldenström Macroglobulinemia.

PURPOSE: p21-activated kinase 4 (PAK4) plays a significant biological and functional role in a number of malignancies, including multiple myeloma (MM). On the basis of our promising findings in MM, we here characterize PAK4 expression and role in WM cells, as well effect of dual PAK4-NAMPT inhibitor (KPT-9274) against WM cell growth and viability. EXPERIMENTAL DESIGN: We have analyzed mRNA and protein expression levels of PAK4 in WM cells, and used loss-of-function approach to investigate its contribution to WM cell viability. We have further tested the in vitro and in vivo effect of KPT-9274 against WM cell growth and viability. RESULTS: We report here high-level expression and functional role of PAK4 in WM, as demonstrated by shRNA-mediated knockdown; and significant impact of KPT-9274 on WM cell growth and viability. The growth inhibitory effect of KPT-9274 was associated with decreased PAK4 expression and NAMPT activity, as well as induction of apoptosis. Interestingly, in WM cell lines treated with KPT-9274, we detected a significant impact on DNA damage and repair genes. Moreover, we observed that apart from inducing DNA damage, KPT-9274 specifically decreased RAD51 and the double-strand break repair by the homologous recombination pathway. As a result, when combined with a DNA alkylating agents bendamustine and melphalan, KPT-9274 provided a synergistic inhibition of cell viability in WM cell lines and primary patient WM cells in vitro and in vivo. CONCLUSIONS: These results support the clinical investigation of KPT-9274 in combination with DNA-damaging agent for treatment of WM.

Role of apurinic/apyrimidinic nucleases in the regulation of homologous recombination in myeloma: mechanisms and translational significance.

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.

Genomic patterns of progression in smoldering multiple myeloma.

We analyzed whole genomes of unique paired samples from smoldering multiple myeloma (SMM) patients progressing to multiple myeloma (MM). We report that the genomic landscape, including mutational profile and structural rearrangements at the smoldering stage is very similar to MM. Paired sample analysis shows two different patterns of progression: a "static progression model", where the subclonal architecture is retained as the disease progressed to MM suggesting that progression solely reflects the time needed to accumulate a sufficient disease burden; and a "spontaneous evolution model", where a change in the subclonal composition is observed. We also observe that activation-induced cytidine deaminase plays a major role in shaping the mutational landscape of early subclinical phases, while progression is driven by APOBEC cytidine deaminases. These results provide a unique insight into myelomagenesis with potential implications for the definition of smoldering disease and timing of treatment initiation.

Phosphatidylserine-exposing blood cells and microparticles induce procoagulant activity in non-valvular atrial fibrillation.

BACKGROUND: The definitive role of phosphatidylserine (PS) in the prothrombotic state of non-valvular atrial fibrillation (NVAF) remains unclear. Our objectives were to study the PS exposure on blood cells and microparticles (MPs) in NVAF, and evaluate their procoagulant activity (PCA). METHODS: NVAF patients without (n = 60) and with left atrial thrombi (n = 18) and controls (n = 36) were included in our study. Exposed PS was analyzed with flow cytometry and confocal microscopy. PCA was evaluated using clotting time, factor Xa (FXa), thrombin and fibrin formation. RESULTS: PS+ blood cells and MPs were significantly higher in NVAF patients without and with left atrial thrombi (both P < 0.01) than in controls. Patients with left atrial thrombi showed increased PS+ platelets, neutrophils, erythrocytes and MPs compared with patients without thrombi (all P < 0.05). Moreover, in patients with left atrial thrombi, MPs primarily originated from platelets (56.1%) followed by leukocytes (21.9%, including MPs from neutrophils, monocytes and lymphocytes), erythrocytes (12.2%) and endothelial cells (8.9%). Additionally, PS+ blood cells and MPs contributed to markedly shortened coagulation time and dramatically increased FXa/thrombin/fibrin (all P < 0.001) generation in both NVAF groups. Furthermore, blockade of exposed PS on blood cells and MPs with lactadherin inhibited PCA by approximately 80%. Lastly, we found that the amount of PS+ platelets and MPs was positively correlated with thrombus diameter (all p < 0.005). CONCLUSIONS: Our results suggest that exposed PS on blood cells and MPs play a procoagulant role in NVAF patients. Blockade of PS prior to thrombus formation might be a novel therapeutic approach in these patients.

Impact of RAD51C-mediated Homologous Recombination on Genomic Integrity in Barrett's Adenocarcinoma Cells.

BACKGROUND: In normal cells, RAD51-mediated homologous recombination (HR) is a precise DNA repair mechanism which plays a key role in the maintenance of genomic integrity and stability. However, elevated (dysregulated) RAD51 is implicated in genomic instability and is a potential target for treatment of certain cancers, including Barrett's adenocarcinoma (BAC). In this study, we investigated genomic impact and translational significance of moderate vs. strong suppression of RAD51 in BAC cells. METHODS: BAC cells (FLO-1 and OE33) were transduced with non-targeting control (CS) or RAD51-specific shRNAs, mediating a moderate (40-50%) suppression or strong (80-near 100%) suppression of the gene. DNA breaks, spontaneous or following exposure to DNA damaging agent, were examined by comet assay and 53BP1 staining. Gene expression was monitored by microarrays (Affymetrix). Homologous recombination (HR) and single strand annealing (SSA) activities were measured using plasmid based assays. RESULTS: We show that although moderate suppression consistenly inhibits/reduces HR activity, the strong suppression is associated with increase in HR activity (by ~15 - ≥ 50% in various experiments), suggesting activation of RAD51-independent pathway. Contrary to moderate suppression, a strong suppression of RAD51 is associated with a significant induced DNA breaks as well as altered expression of genes involved in detection/processing of DNA breaks and apoptosis. Stronger RAD51 suppression was also associated with mutagenic single strand annealing mediated HR. Suppression of RAD51C inhibited RAD51-independent (SSA-mediated) HR in BAC cells. CONCLUSION: Elevated (dysregulated) RAD51 in BAC is implicated in both the repair of DNA breaks as well as ongoing genomic rearrangements. Moderate suppression of this gene reduces HR activity, whereas strong or near complete suppression of this gene activates RAD51C-dependent HR involving a mechanism known as single strand annealing (SSA). SSA-mediated HR, which is a mutagenic HR pathway, further disrupts genomic integrity by increasing DNA breaks in BAC cells.