Investigation of the intrinsic mechanism of drug resistance in multiple myeloma

The focus of this thesis was to evaluate the mechanisms whereby myeloma cells develop intrinsic resistance with a focus on resistance in the context of bortezomib treatment. The aims of this thesis were to examine multidrug resistance pumps as a mechanism of resistance in MM, to investigate the contribution of p53 signalling perturbations in resistance mechanism in MM, to study the AMPK pathway as an alternative target to overcome MM resistance and finally to characterise myeloma resistance to bortezomib treatment using 2D-DIGE analysis. Focussing on bortezomib resistance models, we found that that overexpression of P-gp attenuates bortezomib activity. Bortezomib is a P-gp substrate and a combination of P-gp inhibitor and bortezomib is able to overcome resistance. Bortezomib is also able to downregulate the expression and function of P-gp. Our findings therefore suggest that combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable treatment combination to extend use of the drug. We have shown that p53 apoptotic signalling pathways can be accentuated when bortezomib is combined with a Mdm2 inhibitor. In p53 WT cells, nutlin-3 in combination with bortezomib generates additive toxicity in MM cells but is highly synergistic in epithelial models and p53-mutated cell lines. This synergy persists in the presence of BMSCs. This observation has implications more so in epithelial cancers and p53 mutated cancers where single agent bortezomib activity is mild. We have also shown that bortezomib-treated patients who had high expression of nutlin-3-suppressed genes had significantly shorter progression-free (p=0.001, log-rank test) and overall survival (p=0.002, log-rank test) compared to those with low expression levels. AMPK activation is promising as an anticancer pathway and may also be a chemoprevention target. Metformin and AICAR, which activate this pathway, both have demonstrated useful preclinical anticancer properties and have a good therapeutic index in patients. We explored mechanism of cell death and showed that AICAR was able to activate the apoptotic pathway. These agents also synergise with glycolysis inhibitors to further increase cytotoxicity in cancer cells. Identification of proteins whose expression is altered in differing states of sensitivity and resistance provides candidates for better understanding of resistance mechanisms so we also investigated bortezomib resistance in cellular models using proteomic techniques and isolated and identified several novel proteins which may play a role in this phenomenon. Our findings are mechanistically consistent since two of the identified proteins Hsp70 and caspase-3 are known in the literature to be affected by bortezomib treatment

Healthcare Worker Seroconversion in SARS Outbreak

Serum samples were obtained from healthcare workers 5 weeks after exposure to an outbreak of severe acute respiratory syndrome (SARS). A sensitive dot blot enzyme-linked immunosorbent assay, complemented by a specific neutralization test, shows that only persons in whom probable SARS was diagnosed had specific antibodies and suggests that subclinical SARS is not an important feature of the disease

Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes

The paired-end ditagging (PET) technique has been shown to be efficient and accurate for large-scale transcriptome and genome analysis. However, as with other DNA tag-based sequencing strategies, it is constrained by the current efficiency of Sanger technology. A recently developed multiplex sequencing method (454-sequencing™) using picolitre-scale reactions has achieved a remarkable advance in efficiency, but suffers from short-read lengths, and a lack of paired-end information. To further enhance the efficiency of PET analysis and at the same time overcome the drawbacks of the new sequencing method, we coupled multiplex sequencing with paired-end ditagging (MS-PET) using modified PET procedures to simultaneously sequence 200 000 to 300 000 dimerized PET (diPET) templates, with an output of nearly half-a-million PET sequences in a single 4 h machine run. We demonstrate the utility and robustness of MS-PET by analyzing the transcriptome of human breast carcinoma cells, and by mapping p53 binding sites in the genome of human colorectal carcinoma cells. This combined sequencing strategy achieved an approximate 100-fold efficiency increase over the current standard for PET analysis, and furthermore enables the short-read-length multiplex sequencing procedure to acquire paired-end information from large DNA fragments

ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing

ChIA-PET Tool can be used to process long-range chromatin interaction data. Results are visualized on a user-friendly genome browser

As Omicron Takes Hold and Other New Variants Arise, COVID-19 Testing Remains the Universally Agreed Tool to Effect Transition From Pandemic to Endemic State

The COVID-19 pandemic has caused more than 448 million cases and 6 million deaths worldwide to date. Omicron is now the dominant SARS-CoV-2 variant, making up more than 90% of cases in countries reporting sequencing data. As the pandemic continues into its third year, continued testing is a strategic and necessary tool for transitioning to an endemic state of COVID-19. Here, we address three critical topics pertaining to the transition from pandemic to endemic: defining the endemic state for COVID-19, highlighting the role of SARS-CoV-2 testing as endemicity is approached, and recommending parameters for SARS-CoV-2 testing once endemicity is reached. We argue for an approach that capitalizes on the current public health momentum to increase capacity for PCR-based testing and whole genome sequencing to monitor emerging infectious diseases. Strategic development and utilization of testing, including viral panels in addition to vaccination, can keep SARS-CoV-2 in a manageable endemic state and build a framework of preparedness for the next pandemic

Retroperitoneal liposarcomas: The experience of a tertiary Asian center

10.1186/1477-7819-9-12World Journal of Surgical Oncology9

Microenvironmental Influence on Pre-Clinical Activity of Polo-Like Kinase Inhibition in Multiple Myeloma: Implications for Clinical Translation

Polo-like kinases (PLKs) play an important role in cell cycle progression, checkpoint control and mitosis. The high mitotic index and chromosomal instability of advanced cancers suggest that PLK inhibitors may be an attractive therapeutic option for presently incurable advanced neoplasias with systemic involvement, such as multiple myeloma (MM). We studied the PLK 1, 2, 3 inhibitor BI 2536 and observed potent (IC50<40 nM) and rapid (commitment to cell death <24 hrs) in vitro activity against MM cells in isolation, as well as in vivo activity against a traditional subcutaneous xenograft mouse model. Tumor cells in MM patients, however, don't exist in isolation, but reside in and interact with the bone microenvironment. Therefore conventional in vitro and in vivo preclinical assays don't take into account how interactions between MM cells and the bone microenvironment can potentially confer drug resistance. To probe this question, we performed tumor cell compartment-specific bioluminescence imaging assays to compare the preclinical anti-MM activity of BI 2536 in vitro in the presence vs. absence of stromal cells or osteoclasts. We observed that the presence of these bone marrow non-malignant cells led to decreased anti-MM activity of BI 2536. We further validated these results in an orthotopic in vivo mouse model of diffuse MM bone lesions where tumor cells interact with non-malignant cells of the bone microenvironment. We again observed that BI 2536 had decreased activity in this in vivo model of tumor-bone microenvironment interactions highlighting that, despite BI 2536's promising activity in conventional assays, its lack of activity in microenvironmental models raises concerns for its clinical development for MM. More broadly, preclinical drug testing in the absence of relevant tumor microenvironment interactions may overestimate potential clinical activity, thus explaining at least in part the gap between preclinical vs. clinical efficacy in MM and other cancers

Functional annotation of human long noncoding RNAs via molecular phenotyping

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-todate lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.Peer reviewe