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.

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

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 TOP2ß. 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.

Production of immunogenic human papillomavirus-16 major capsid protein derived virus like particles.

BACKGROUND & OBJECTIVE: Recombinant DNA technology allows expression of the human papillomavirus (HPV) major capsid protein (L1) in heterologous expression systems and the recombinant protein self assembles to virus-like particles (VLP). We took up this study to produce recombinant HPV-16 L1 in yeast, establish the process of recombinant L1 derived VLP preparation and develop an ELISA using VLP as the antigen for serological evaluation of anti HPV-16 L1 antibody status. METHODS: Complete HPV-16 L1 was amplified from genomic DNA of an esophageal cancer biopsy, cloned and the protein was expressed in a galactose-inducible Saccharomyces cerevisiae expression system. Self assembled VLP was purified by a two-step density gradient centrifugation process and the VLP preparation used to test its suitability in developing an ELISA. RESULTS: The recombinant protein was predominantly a ~55 KD species with distinct immunoreactivity and formed VLP as confirmed by electron microscopy. An ELISA using the VLP showed its efficacy in appropriate immunoreactivity to serum/plasma IgG. INTERPRETATION & CONCLUSION: Recombinant HPV-16 capsid protein derived VLP was produced and the VLP antigen based ELISA can be used to probe serological association of HPV with different clinical conditions. The VLP technology can be improved further and harnessed for future vaccine development efforts in the country.