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.

Molecular Crosstalk between Chromatin Remodeling and Tumor Microenvironment in Multiple Myeloma.

Multiple myeloma (MM) is a complex disease driven by numerous genetic and epigenetic alterations that are acquired over time. Despite recent progress in the understanding of MM pathobiology and the availability of innovative drugs, which have pronounced clinical outcome, this malignancy eventually progresses to a drug-resistant lethal stage and, thus, novel therapeutic drugs/models always play an important role in effective management of MM. Modulation of tumor microenvironment is one of the hallmarks of cancer biology, including MM, which affects the myeloma genomic architecture and disease progression subtly through chromatin modifications. The bone marrow niche has a prime role in progression, survival, and drug resistance of multiple myeloma cells. Therefore, it is important to develop means for targeting the ecosystem between multiple myeloma bone marrow microenvironment and chromatin remodeling. Extensive gene expression profile analysis has indeed provided the framework for new risk stratification of MM patients and identifying novel molecular targets and therapeutics. However, key tumor microenvironment factors/immune cells and their interactions with chromatin remodeling complex proteins that drive MM cell growth and progression remain grossly undefined.

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.

YWHAE/14-3-3ε expression impacts the protein load, contributing to proteasome inhibitor sensitivity in multiple myeloma.

High protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.

Deregulation of LIMD1-VHL-HIF-1α-VEGF pathway is associated with different stages of cervical cancer.

To understand the mechanism of cellular stress in basal-parabasal layers of normal cervical epithelium and during different stages of cervical carcinoma, we analyzed the alterations (expression/methylation/copy number variation/mutation) of HIF-1α and its associated genes LIMD1, VHL and VEGF in disease-free normal cervix (n = 9), adjacent normal cervix of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 32), cancer of uterine cervix (CACX; n = 174) samples and two CACX cell lines. In basal-parabasal layers of normal cervical epithelium, LIMD1 showed high protein expression, while low protein expression of VHL was concordant with high expression of HIF-1α and VEGF irrespective of HPV-16 (human papillomavirus 16) infection. This was in concordance with the low promoter methylation of LIMD1 and high in VHL in the basal-parabasal layers of normal cervix. LIMD1 expression was significantly reduced while VHL expression was unchanged during different stages of cervical carcinoma. This was in concordance with their frequent methylation during different stages of this tumor. In different stages of cervical carcinoma, the expression pattern of HIF-1α and VEGF was high as seen in basal-parabasal layers and inversely correlated with the expression of LIMD1 and VHL. This was validated by demethylation experiments using 5-aza-2'-deoxycytidine in CACX cell lines. Additional deletion of LIMD1 and VHL in CIN/CACX provided an additional growth advantage during cervical carcinogenesis through reduced expression of genes and associated with poor prognosis of patients. Our data showed that overexpression of HIF-1α and its target gene VEGF in the basal-parabasal layers of normal cervix was due to frequent inactivation of VHL by its promoter methylation. This profile was maintained during different stages of cervical carcinoma with additional methylation/deletion of VHL and LIMD1.

Activation of Wnt-ß-catenin pathway in basal-parabasal layers of normal cervical epithelium comparable during development of uterine cervical carcinoma.

In this study, importance of Wnt-ß-catenin pathway in the development of uterine cervical carcinoma was evaluated. For this purpose, the profiles (expression/methylation/deletion) of ß-catenin, p-ß-catenin (Y654), Wnt3a, and APC were studied in disease free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of primary tumors (n = 70), CIN (n = 28), CACX (n = 102) samples, and two CACX cell lines (HeLa and SiHa). Immunohistochemical analysis revealed high/medium (74-95%) expression of ß-catenin/p-ß-catenin (Y654) and Wnt3a and low expression (23-26%) of APC in proliferating basal-parabasal layers contrary to differentiated spinous layer in normal cervix irrespective of HPV16 infection. The expression profile of the genes in the basal-parabasal layers did not change significantly during development of CACX. High (66%) promoter methylation of APC was seen in basal-parabasal layers and the cervical lesions (42-69%), unlike in spinous layers (25%). The promoter methylation status of APC was validated by in vitro demethylation experiments using 5-aza-dC in CACX cell lines. However, additional deletion of APC was significantly increased from CIN (12%) to stage I/II (40%) and became comparable in stage III/IV (48%) of the tumor. Patients with alterations (deletion/methylation) of APC and high/medium expression of Wnt3a/ß-catenin/p-ß-catenin (Y654) showed significantly poor survival. Thus our data indicate that cumulative effect of Wnt3a overexpression and APC inactivation are needed for overexpression of ß-catenin during the development of CACX.

Integrative genomic and network analysis identified novel genes associated with the development of advanced cervical squamous cell carcinoma.

BACKGROUND: CSCC is one of the most common cancer affecting women globally. Though it is caused by the infection of hrHPV but long latency period for malignant outcome in only a subset of hrHPV infected women indicates involvement of additional alterations, primarily CNVs. Here, we showed how CNVs played a crucial role in development of advanced tumors (stage III/IV) in Indian patients. METHODS: Initially, high-resolution CGH-SNP microarray analysis pointed out frequent CNVs followed by significantly altered genes. After comparison with TCGA dataset, expressions of the genes were checked in three CSCC datasets to identify key genes followed by Ingenuity® Pathway analysis. Then node effect property analysis was applied on the constructed PPI network to rank the key proteins. Finally, validations in independent samples were performed. RESULTS: For the first time, frequent chromosomal amplifications at 3q13.13-3q29, 1p36.11-1p31.1, 1q21.1-1q44 and 5p15.33-5p12 followed by common deletions at 11q14.1-11q25, 2q34-2q37.3, 4p16.3-4p12 and 13q13.3-13q14.3 were identified in Indian CSCC patients. Integrative analysis found 78 key genes including several novel ones, which were mostly associated with 'Cancer' and may regulate DNA repair and metabolic pathways. Analysis showed PARP1 and ATR were among the top ranking protein interactors. CONCLUSIONS: Frequent amplification and over-expression of ATR and PARP1 were further confirmed in cervical lesions, indicating their association with poor prognosis of advanced CSCC patients. GENERAL SIGNIFICANCE: Our novel approach identified precise CNVs along with several novel genes within these loci and showed that PARP1 and ATR, having biologically significant interactions, may be involved in development of advanced CSCC.

Association of P16-RBSP3 inactivation with phosphorylated RB1 overexpression in basal-parabasal layers of normal cervix unchanged during CACX development.

To understand the molecular mechanism of RB1 phosphorylation in basal-parabasal layers of normal cervix and during cervical cancer (CACX) development, we analyzed the alterations (expression/methylation/deletion/mutation) of RB1/phosphorylated RB1 (p-RB1) (ser807/811 and ser567) and two RB1 phosphorylation inhibitors, P16 and RBSP3, in disease-free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 28), CACX (n = 102) samples and two CACX cell lines. Immunohistochemical analysis revealed high/medium expression of RB1/p-RB1 (ser807/811 and ser567) and low expression of P16 and RBSP3 in proliferating basal-parabasal layers of majority of normal cervical epitheliums, irrespective of HPV16 infection. Interestingly, 35-52% samples showed high/medium expression of P16 in basal-parabasal layers of normal and had significant association with deleterious non-synonimous SNPs of P16. Methylation of P16 and RBSP3 in basal-parabasal layers of normal cervix (32 and 62%, respectively) showed concordance with their respective expressions in basal-parabasal layers. The methylation frequency of P16 and RBSP3 in basal-parabasal layers of normal did not change significantly in CIN and CACX. The deletion frequency of P16 and RB1 increased significantly with CACX progression. While, deletion of RBSP3 was high in CIN and comparable during CACX progression. P16 showed scattered and infrequent mutation in CACX. The alteration of P16 and RBSP3 was synergistic and showed association with overexpression of p-RB1 in tumors and associated with poor prognosis of patients. Thus, our data suggest that overexpression of p-RB1 in basal-parabasal layers of normal cervical epithelium was due to methylation/low functional-linked non-synonimous SNPs of P16 and RBSP3. This pattern was maintained during cervical carcinogenesis by additional deletion/mutation.

Persistent HPV16/18 infection in Indian women with the A-allele (rs6457617) of HLA-DQB1 and T-allele (rs16944) of IL-1ß -511 is associated with development of cervical carcinoma.

The aim of this study was to understand the association of human papillomavirus (HPV) type 16/18 infection and polymorphisms in the HLA-DQB1 (rs6457617) and IL-1ß -511 (rs16944) loci with the development of uterine cervical cancer (CaCx). The distribution of HLA-DQB1 G > A and IL-1ß -511 C/T polymorphisms was determined in HPV-negative cervical swabs from normal women (N = 111) and compared with cervical swabs of HPV-cleared normal women (once HPV infected followed by natural clearance of the infection, N = 86), HPV16/18-positive cervical intraepithelial neoplasia (CIN, N = 41) and CaCx biopsies (N = 107). The A-allele containing genotypes (i.e. G/A and A/A) of HLA-DQB1 was significantly associated with CaCx compared with HPV-negative [OR = 2.56(1.42-4.62), p = 0.001] or HPV-cleared [OR = 2.07(1.12-3.87), p = 0.01] normal women, whereas the T-allele containing genotypes (i.e. C/T and T/T) of IL-1ß showed increased risk of CIN [OR = 3.68(0.97-16.35), p = 0.03; OR = 3.59(0.92-16.38), p = 0.03] and CaCx development [OR = 2.03(1.03-5.2), p = 0.02; OR = 2.25(0.96-5.31), p = 0.04] compared with HPV-negative or HPV-cleared normal women. Considering these two loci together, it was evident that the T- and A-alleles rendered significantly increased susceptibility for development of CIN and CaCx compared with HPV-negative and HPV-cleared normal women. Moreover, the T-allele of IL-1ß showed increased susceptibility for CIN [OR = 3.62(0.85-17.95), p = 0.04] and CaCx [OR = 2.39(0.91-6.37), p = 0.05] development compared with the HPV-cleared women, even in the presence of the HLA-DQB1 G-allele. Thus, our data suggest that persistent HPV16/18 infection in the cervix due to the presence of the HLA-DQB1 A-allele and chronic inflammation due to the presence of the IL-1ß -511 T-allele might predispose women to CaCx development.

Physical and methylation status of human papillomavirus 16 in asymptomatic cervical infections changes with malignant transformation.

AIMS: To evaluate how the genetic and epigenetic profile of human papillomavirus 16 (HPV16) changes from asymptomatic cervical infections to cervical cancer (CaCx) development. METHODS: HPV16 physical status, methylation of its early-late promoters and its upstream enhancer sequences were analysed in samples from asymptomatic cervical infections (n=89), pre-neoplastic lesions (low and high grade squamous intraepithelial lesions LSIL/HSIL, n=28) and primary CaCx (n=98). RESULTS: In asymptomatic infection (65%, 58/89) and LSIL/HSIL (57%, 16/28) samples, the episomal form of HPV16 was predominant whereas integration of HPV16 was significantly (p=0.01) higher in CaCx (59%, 57/98). The integrated viral form was also present in asymptomatic (27%, 24/89) and LSIL/HSIL (25%, 7/28) samples. The methylation of the enhancer region was comparable (29-34%) among asymptomatic, LSIL/HSIL and CaCx samples. The episomal form exhibited relatively higher methylation of the early promoter (52%) than that of the late promoter (40%) in asymptomatic infection but the integrated form in asymptomatic carriers showed the opposite methylation pattern (early promoter (42%) vs late-promoter (54%)). A similar pattern was observed in LSIL/HSIL samples, with comparable frequencies (44%) of early and late promoter methylation of the episomal form. However, irrespective of HPV16 physical status, higher methylation of late promoter than that of early promoter was observed in CaCx samples. An inverse correlation was observed between HPV16 integration and overall methylation of the early promoter-enhancer region in CaCx (p=0.05), LSIL/HSIL (p=0.09) and asymptomatic samples (p=0.09). CONCLUSIONS: Our study indicates that integration of HPV16 along with changes in methylation pattern of early and late promoters is essential for neoplastic transformation of asymptomatic cervical infections.

Inactivation of PTCH1 is associated with the development of cervical carcinoma: clinical and prognostic implication.

The aim of this study was to analyze the alterations of PTCH1 (deletion/promoter methylation/mutation/expression) during the development of cervical cancer (CACX). For this purpose, deletion/methylation of PTCH1 were analyzed in HPV16 positive exfoliated asymptomatic cervical swabs (n = 74), cervical intraepithelial neoplasia (CIN) (n = 32), CACX (n = 174) samples, and two CACX cell lines. The deletion of PTCH1 increased significantly from CIN (11.5%) to stage I/II (42%) and comparable in stage III/IV (46%). Low frequency (14-16%) of PTCH1 methylation was seen in the asymptomatic exfoliated cervical cells and in the normal epithelium adjacent to the tumor followed by a significant increase in CIN (31%) to stage I/II (57%) and comparable in stage III/IV (58%). The overall alterations (deletion/methylation) of PTCH1 significantly increased from CIN (34%) to stage I/II (70%) and comparable in stage III/IV (69%). Interestingly, in the normal epithelium, methylation of PTCH1 was high in basal/parabasal layers (83%), followed by decrease in the spinous layer (33 %), and showed significant inverse correlation with its expression. Reduced expression of PTCH1 seen in tumors showed a significant association with its alterations (deletion/methylation). The expression pattern of PTCH1 showed an inverse correlation with the nuclear expression of GLI1 in the normal epithelium as well as in the tumors. High nuclear expression of HPV16, E6, and E7 were seen in basal/parabasal layers of the normal epithelium and also in tumors. The PTCH1 alterations (deletion and/or methylation) in tumors and its methylation in adjacent normal epithelium were associated with poor prognosis of patients. Thus, our data suggests that activation of the Hedgehog pathway due to PTCH1 inactivation along with HPV infection is important in CACX development.

Genetic and epigenetic changes of HPV16 in cervical cancer differentially regulate E6/E7 expression and associate with disease progression.

OBJECTIVE: The study was aimed at understanding the complex interactions of genetic and epigenetic events in expression of HPV16 E6/E7 and progression of cervical carcinoma. For this, expression of E6/E7 was done in 36 samples, along with the physical status, methylation and LCR sequence variations. Later, the genetic and epigenetic studies were extended to 239 samples to find out the association of these factors with progression of cervical cancer. METHODS: E6/E7 expression was quantified by real-time PCR. Physical status of HPV16 was determined by mutiplex-PCR of whole E2 ORF using overlapping primers and E6 ORF and validated by real-time PCR. Methylation status of P97 promoter/enhancer was analyzed by methylation sensitive restriction analysis (MSRA). Viral lineage and variations in LCR was ascertained by sequencing LCR/E6/E7 ORFs. RESULTS: Samples with episomal unmethylated virus showed comparatively high expression of E6/E7 than episomal methylated, integrated unmethylated and integrated methylated forms of HPV16. Variations in the LCR, particularly in the binding sites of negatively regulating transcription factors, also contribute to high expression of E6/E7. The integrated form significantly increases with decrease of episomal form during tumor progression. Methylation of the promoter/enhancer gradually decreased with tumor progression and is inversely correlated to integration. Two novel variants were observed in E6 gene in European- and North-American-1-lineages. Log-rank test revealed better prognosis of the patients with episomal methylated HPV16 compared to the other forms. CONCLUSION: Our results show higher expression of E6/E7 in samples with episomal unmethylated virus having sequence variations in LCR.