Ekjot Kaur

Radiation-induced homotypic cell fusions of innately resistant glioblastoma cells mediate their sustained survival and recurrence

Understanding of molecular events underlying resistance and relapse in glioblastoma (GBM) is hampered due to lack of accessibility to resistant cells from patients undergone therapy. Therefore, we mimicked clinical scenario in an in vitro cellular model developed from five GBM grade IV primary patient samples and two cell lines. We show that upon exposure to lethal dose of radiation, a subpopulation of GBM cells, innately resistant to radiation, survive and transiently arrest in G2/M phase via inhibitory pCdk1(Y15). Although arrested, these cells show multinucleated and giant cell phenotype (MNGC). Significantly, we demonstrate that these MNGCs are not pre-existing giant cells from parent population but formed via radiation-induced homotypic cell fusions among resistant cells. Furthermore, cell fusions induce senescence, high expression of senescence-associated secretory proteins (SASPs) and activation of pro-survival signals (pAKT, BIRC3 and Bcl-xL) in MNGCs. Importantly, following transient non-proliferation, MNGCs escape senescence and despite having multiple spindle poles during mitosis, they overcome mitotic catastrophe to undergo normal cytokinesis forming mononucleated relapse population. This is the first report showing radiation-induced homotypic cell fusions as novel non-genetic mechanism in radiation-resistant cells to sustain survival. These data also underscore the importance of non-proliferative phase in resistant glioma cells. Accordingly, we show that pushing resistant cells into premature mitosis by Wee1 kinase inhibitor prevents pCdk1(Y15)-mediated cell cycle arrest and relapse. Taken together, our data provide novel molecular insights into a multistep process of radiation survival and relapse in GBM that can be exploited for therapeutic interventions.

Clinical implications of MTA proteins in human cancer

Metastasis-associated gene or metastasis tumor antigen 1 (MTA1) is a new member of cancer progression-related gene family. It was first identified in rat mammary adenocarcinoma and later recognized as an important constituent of nucleosomal remodeling complex (NuRD), displaying dual regulatory functions as a co-repressor and co-activator for a large number of genes. Chromatin remodelers are ATP-dependent multi-protein chromatin modifying machines. These complexes alter the nucleosome positioning regulating the accessibility of genomic DNA to various transcription factors and thus modulate eukaryotic gene transcription. Since its identification two decades ago, MTA1 has been reported to be overexpressed in many cancers. Moreover, its overexpression has also been correlated with transformation and tumor progression. Furthermore, MTA1 has been shown to modulate the response of several tumor suppressor genes like p53 and oncogenes like c-myc. Taken together, current literature suggests that MTA proteins, especially MTA1, act as a master co-regulatory molecule involved in the carcinogenesis and progression of various malignant tumors. The primary focus of this review is to provide an overview of the MTA proteins with special emphasis on its role in cancer and use as a marker for cancer progression and potential target for therapy.

Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer

Background Notch pathway plays a complex role depending on cellular contexts: promotes stem cell maintenance or induces terminal differentiation in potential cancer-initiating cells; acts as an oncogene in lymphocytes and mammary tissue or plays a growth-suppressive role in leukemia, liver, skin, and head and neck cancer. Here, we present a novel clinical and functional significance of NOTCH1 alterations in early stage tongue squamous cell carcinoma (TSCC). Patients and Methods We analyzed the Notch signaling pathway in 68 early stage TSCC primary tumor samples by whole exome and transcriptome sequencing, real-time PCR based copy number, expression, immuno-histochemical, followed by cell based biochemical and functional assays. Results We show, unlike TCGA HNSCC data set, NOTCH1 harbors significantly lower frequency of inactivating mutations (4%); is somatically amplified; and, overexpressed in 31% and 37% of early stage TSCC patients, respectively. HNSCC cell lines over expressing NOTCH1, when plated in the absence of attachment, are enriched in stem cell markers and form spheroids. Furthermore, we show that inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 inhibits spheroid forming capacity, transformation, survival and migration of the HNSCC cells suggesting an oncogenic role of NOTCH1 in TSCC. Clinically, Notch pathway activation is higher in tumors of non-smokers compared to smokers (50% Vs 18%, respectively, P=0.026) and is also associated with greater nodal positivity compared to its non-activation (93% Vs 64%, respectively, P=0.029). Conclusion We anticipate that these results could form the basis for therapeutic targeting of NOTCH1 in tongue cancer.

Unique spectral markers discern recurrent Glioblastoma cells from heterogeneous parent population.

An inability to discern resistant cells from bulk tumour cell population contributes to poor prognosis in Glioblastoma. Here, we compared parent and recurrent cells generated from patient derived primary cultures and cell lines to identify their unique molecular hallmarks. Although morphologically similar, parent and recurrent cells from different samples showed variable biological properties like proliferation and radiation resistance. However, total RNA-sequencing revealed transcriptional landscape unique to parent and recurrent populations. These data suggest that global molecular differences but not individual biological phenotype could differentiate parent and recurrent cells. We demonstrate that Raman Spectroscopy a label-free, non-invasive technique, yields global information about biochemical milieu of recurrent and parent cells thus, classifying them into distinct clusters based on Principal-Component-Analysis and Principal-Component-Linear-Discriminant-Analysis. Additionally, higher lipid related spectral peaks were observed in recurrent population. Importantly, Raman spectroscopic analysis could further classify an independent set of naïve primary glioblastoma tumour tissues into non-responder and responder groups. Interestingly, spectral features from the non-responder patient samples show a considerable overlap with the in-vitro generated recurrent cells suggesting their similar biological behaviour. This feasibility study necessitates analysis of a larger cohort of naïve primary glioblastoma samples to fully envisage clinical utility of Raman spectroscopy in predicting therapeutic response.

Inhibition of novel GCN5-ATM axis restricts the onset of acquired drug resistance in leukemia: GCN5-ATM axis in leukemia resistance

Leukemia is majorly treated by topoisomerase inhibitors that induce DNA double strand breaks (DSB) resulting in cell death. Consequently, modulation of DSB repair pathway renders leukemic cells resistant to therapy. As we do not fully understand the regulation of DSB repair acquired by resistant cells, targeting these cells has been a challenge. Here we investigated the regulation of DSB repair pathway in early drug resistant population (EDRP) and late drug resistant population (LDRP). We found that doxorubicin induced equal DSBs in parent and EDRP cells; however, cell death is induced only in the parent cells. Further analysis revealed that EDRP cells acquire relaxed chromatin via upregulation of lysine acetyl transferase KAT2A (GCN5). Drug treatment induces GCN5 interaction with ATM facilitating its recruitment to DSB sites. Hyperactivated ATM maximize H2AX, NBS1, BRCA1, Chk2, and Mcl‐1 activation, accelerating DNA repair and survival of EDRP cells. Consequently, inhibition of GCN5 significantly reduces ATM activation and survival of EDRP cells. Contrary to EDRP, doxorubicin failed to induce DSBs in LDRP because of reduced drug uptake and downregulation of TOP2β. Accordingly, ATM inhibition prior to doxorubicin treatment completely eliminated EDRP but not LDRP. Furthermore, baseline AML samples (n = 44) showed significantly higher GCN5 at mRNA and protein levels in MRD positive compared to MRD negative samples. Additionally, meta‐analysis (n = 221) showed high GCN5 expression correlates with poor overall survival. Together, these results provide important insights into the molecular mechanism specific to EDRP and will have implications for the development of novel therapeutics for AML.

Enhanced proteasomal activity is essential for long term survival and recurrence of innately radiation resistant residual glioblastoma cells

Therapy resistance and recurrence in Glioblastoma is due to the presence of residual radiation resistant cells. However, because of their inaccessibility from patient biopsies, the molecular mechanisms driving their survival remain unexplored. Residual Radiation Resistant (RR) and Relapse (R) cells were captured using cellular radiation resistant model generated from patient derived primary cultures and cell lines. iTRAQ based quantitative proteomics was performed to identify pathways unique to RR cells followed by in vitro and in vivo experiments showing their role in radio-resistance. 2720 proteins were identified across Parent (P), RR and R population with 824 and 874 differential proteins in RR and R cells. Unsupervised clustering showed proteasome pathway as the most significantly deregulated pathway in RR cells. Concordantly, the RR cells displayed enhanced expression and activity of proteasome subunits, which triggered NFkB signalling. Pharmacological inhibition of proteasome activity led to impeded NFkB transcriptional activity, radio-sensitization of RR cells in vitro, and significantly reduced capacity to form orthotopic tumours in vivo. We demonstrate that combination of proteasome inhibitor with radio-therapy abolish the inaccessible residual resistant cells thereby preventing GBM recurrence. Furthermore, we identified first proteomic signature of RR cells that can be exploited for GBM therapeutics.

Molecular features unique to glioblastoma radiation resistant residual cells may affect patient outcome - a short report

PurposePreviously we have shown, using a primary glioblastoma (GBM) cell model, that a subpopulation of innately radiation resistant (RR) GBM cells survive radiotherapy and form multinucleated and giant cells (MNGCs) by homotypic fusions. We also showed that MNGCs may cause relapse. Here, we set out to explore whether molecular characteristics of RR cells captured from patient-derived primary GBM cultures bear clinical relevance.MethodsPrimary cultures were derived from 19 naive GBM tumor samples. RR cells generated from these cultures were characterized using various cell biological assays. We also collected clinicopathological data of the 19 patients and assessed associations with RR variables using Spearman’s correlation test and with patient survival using Kaplan-Meier analysis. Significance was determined using a log-rank test.ResultsWe found that SF2 (surviving fraction 2) values (p = 0.029), days of RR cell formation (p = 0.019) and percentage of giant cells (p = 0.034) in the RR population independently correlated with a poor patient survival. We also found that low ATM (Ataxia-telangiectasia mutated) expression levels in RR cells showed a significant (p = 0.002) negative correlation with SF2 values. A low ATM expression level in RR cells along with a high tumor volume was also found to negatively correlate with patient survival (p = 0.011). Finally, we found that the ATM expression levels in RR cells independently correlated with a poor patient survival (p = 0.014).ConclusionsOur data indicate that molecular features of innately radiation resistant GBM cells independently correlate with clinical outcome. Our study also highlights the relevance of using patient-derived primary GBM cultures for the characterization of RR cells that are otherwise inaccessible for analysis.

Abstract 5845: Identification of proteosome pathway and a novel serine threonine kinase DCLK3: Potential therapeutic targets for innately radiation resistant glioblastoma cells

INTRODUCTION Glioblastoma resistance and recurrence is attributed to the presence of innately Radiation Resistant (RR) cells present in the heterogeneous parent tumour. However, targeting these cells has been impossible due to inaccessibility of these cells. METHODOLOGY We therefore recapitulated clinical scenario of resistance in a cellular model developed from fresh primary GBM patient samples and cell lines. The model allowed us to capture 1) Parent cells 2) innately Radiation Resistant cells - less than 10% of the parent population and 3) Relapse (R) cells. To identify the targetable proteins governing the survival of RR cells, we performed iTRAQ based quantitative proteomic analysis on all the three populations from GBM cell line (SF 268). RESULTS The proteomic data analysis identified 34 proteins as differentially present in RR population of which 22 were upregulated and 12 were downregulated. A GENE STRING analysis of all the differential proteins in RR population revealed putative interaction of a novel serine threonine kinase DCLK3 with 14-3-3 zeta. The increased expression of DCLK3 and 14-3-3 zeta was confirmed by western blot in RR cells of two GBM cell lines and 8 patient samples. Meta-analysis of 242 tumor samples from COSMIC database showed DCLK3 overexpression in 232 tumors. Furthermore, it harbours 8 missense deleterious mutations, 6 of which were in the kinase domain, indicating towards an important kinase function of this protein. We hypothesized that DCLK3 mediated interaction and phosphorylation of 14-3-3 zeta modulated 14-3-3 zeta functions facilitating RR cell survival. For this, first we wanted to see if DCLK3 and 14-3-3 zeta can interact together. In silico docking of DCLK3 with 14-3-3 zeta did show interaction of 14-3-3 zeta and the kinase domain of DCLK3 kinase. This interaction was confirmed by in vitro immunoprecipitation studies. Further studies are ongoing to understand the importance of these proteins and their interaction in GBM recurrence. Additionally, pathway analysis of differentially upregulated proteins in RR cells revealed deregulation of the proteins involved in Ubiquitin-Proteasome System. Indeed, proteasome activity assay showed increased proteosome activity in the RR population of GBM cell lines and Patient samples. Accordingly, Bortezomib, a proteosome inhibitor induced significant apoptosis in the RR population at a concentration significantly lower than that required for inducing apoptosis in the parent cells. SIGNIFICANCE In conclusion this is the first study to identify a proteome signature of innately radiation resistant cells of GBM and identify proteosome pathway and a novel serine-threonine kinase DCLK3 in RR cells as a potential therapeutic target to inhibit GBM radioresistance and recurrence. Citation Format: Jacinth Rajendra, Keshava Datta, , Sheikh Burhan Ud Din Farooqee, Raja Reddy, Nilesh Gardi, Ekjot Kaur, Ketaki Patkar, Aliasgar Moiyadi, Prasanna Venkataraman, Kakoli Bose, Amit Dutt, Harsha Gowda, Shilpee Dutt. Identification of proteosome pathway and a novel serine threonine kinase DCLK3: Potential therapeutic targets for innately radiation resistant glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5845. doi:10.1158/1538-7445.AM2017-5845

Abstract 3174: GCN5 regulates ATM mediated DNA repair responsible for onset of acquired resistance in leukemia

Abstract Introduction- Resistance to therapeutics targeting topoisomerase 2 is a major problem in the treatment of leukemia. Cells which survive and give rise to relapsed leukemia are known to modulate different pathways like downregulation of drug target, reduced drug accumulation, and improved DNA repair; ultimately leading to survival of drug resistant cells. Here, we wanted to identify the earliest detectable changes occurring when cells become resistant to topoisomerase 2 inhibitors. Methodology- We generated early and late stage drug (doxorubicin) resistant leukemic sub cell line from K562 and THP-1 parent cells, HL-60/MX2 resistant sub cell line of HL60 was also used for this study. Molecular changes were analyzed by electron microscopy, quantitative PCR, western blotting, immunofluorescence, flow cytometry, molecular inhibitors and MTT assay. Results were further confirmed in blast cells of AML (n=44) patient samples collected for this study. Furthermore, meta-analysis for survival was done from microarray expression data of 221 patient samples using PrognoScan. Results- We mapped molecular changes acquired by leukemic cells during evolution from Early Drug Resistant Population (EDRP) to Late Drug Resistant Population (LDRP). We found unlike LDRP, EDRP cells do not possess known bona fide drug-resistance mechanisms namely limited drug accumulation, reduced DNA damage or expression of drug target. Instead they survive by acquiring ‘poised epigenetic state’ that enhanced their DNA repair. Mechanistically, GCN5, a histone acetyl transferase get selectively upregulated in EDRP cells mediating higher H4K16 acetylation levels and consequent euchromatin state of EDRP. Upon Doxorubicin treatment, H4K16ac facilitate higher ATM recruitment and activation causing efficient activation of H2AX, NBS1, BRCA1, Chk2 and Mcl-1, accelerating DNA repair and survival of EDRP cells. Inhibition of GCN5 with Doxorubicin treatment significantly reduces H4K16ac levels, ATM recruitment and cell survival of EDRP cells. Similarly, ATM inhibition along with Doxorubicin completely ablates EDRP but not LDRP. Furthermore, baseline AML patient samples (n=44) showed significantly higher GCN5 expression in MRD positive compared to MRD negative samples. Additionally, meta-analysis of 221 AML patients showed increased GCN5 expression associates with poor survival of AML patients. Conclusion- We identify GCN5 expression as marker that defines onset of resistance in leukemia and GCN5 mediated ATM activation via H4K16ac as a novel non-genetic route facilitating EDRP cell survival with enhanced DNA repair. These data also highlight the clinical relevance of targeting GCN5 and ATM during early resistance to prevent emergence of difficult to treat stable diverse resistance in leukemia. Citation Format: Sameer J. Salunkhe, Jyothi Nair, Ekjot Kaur, Neha Chaoudhary, Sanket Shah, Ketaki Patkar, Dev Anand, Navin Khattry, Syed K. Hasan, Shilpee Dutt. GCN5 regulates ATM mediated DNA repair responsible for onset of acquired resistance in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3174. doi:10.1158/1538-7445.AM2017-3174

Abstract 3848: Subtype-specific DNA damage dependent and independent functions of damage repair proteins confer chemo-resistance to leukemia

Resistance to chemotherapy is a major challenge in the field of leukemia. Since, cancer cells can acquire resistance to DNA damage based therapy by modulating DNA Damage Repair (DDR) pathways, an in-depth understanding of DDR is important for novel drug discoveries. Hence, to understand relation between DDR and resistance we used doxorubicin resistant sub-line (K562-R) of K562 and Mitoxantrone resistant sub-line (HL60/MX) of HL60 as a model system. We show that lethal doses of Mitoxantrone to HL60 and HL60/MX do not induce DNA DSBs (double strand breaks) and accordingly there is no activation of Chk2, although there is equal uptake of drug in both, sensitive and resistant cell types. Since Mitoxantrone produce DSBs by targeting TOP2B, we checked the expression of TOPB2 and found significant decrease in the levels of topoisomerase 2 B (TOP2B). Moreover, negative regulator of TOPB2, mir 21-5P is upregulation in untreated HL60/MX explaining the absence of DSBs in HL-60/MX with Mitoxantrone treatment. Surprisingly, although there are no DSBs induced, we find high levels of DNA-PKcs, MRE11 and Rad50 in HL-60/MX. Furthermore, inhibition of DNA-PKcs re-sensitizes HL-60/MX cells to Mitoxantrone, suggesting a DNA damage independent role for DNA-PK in HL-60/MX cells. Recently DNA-PKcs is shown to play key role in fat metabolism. Indeed we find significant differences in the metabolism of HL60 and HL-60/MX where HL-60 cells use glucose but HL-60/MX cells show reduced glucose and glutamine metabolism and an over expression of fatty acid synthase. However link between DNA-PKcs over expression and differential metabolism in these cells still need to be explored. Interestingly, in contrast to HL60 model, Doxorubicin does induce equal DSBs in K562 and K562-R cells. However K562-R cells hyperactivate ATM and Chk2 to repair their DNA and survive. Importantly, we show that K562 start to show elevated activation of ATM and Chk-2 as early as two rounds of drug treatment. Accordingly, ATM inhibition leads to induction of apoptosis in resistant K562 cells. In conclusion, we show that K562 and HL60 cells use the DNA double strand break repair proteins in DNA damage dependent and independent pathways to acquire resistance to DSB inducing chemo therapeutics. Our data also highlight the importance of considering subtype specific therapeutics in leukemia. Note: This abstract was not presented at the meeting. Citation Format: Sameer Salunkhe, Ekjot Kaur, Ashwin Ramaswamy, Ketaki Patkar, Shilpee Dutt. Subtype-specific DNA damage dependent and independent functions of damage repair proteins confer chemo-resistance to leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3848. doi:10.1158/1538-7445.AM2015-3848