Polyphosphate kinase is involved in stress-induced mprAB-sigE-rel signalling in Mycobacteria

Polyphosphate kinase 1 (PPK1) helps bacteria to survive under stress. The ppk1 gene of Mycobacterium tuberculosis was overexpressed in Escherichia coli and characterized. Residues R230 and F176, predicted to be present in the head domain of PPK1, were identified as residues critical for polyphosphate (polyP)-synthesizing ability and dimerization of PPK1. A ppk1 knockout mutant of Mycobacterium smegmatis was compromised in its ability to survive under long-term hypoxia. The transcription of the rel gene and the synthesis of the stringent response regulator ppGpp were impaired in the mutant and restored after complementation with ppk1 of M. tuberculosis, providing evidence that PPK1 is required for the stringent response. We present evidence that PPK1 is likely required for mprAB-sigE-rel signalling. σE regulates the transcription of rel, and we hypothesize that under conditions of stress polyP acts as a preferred donor for MprB-mediated phosphorylation of MprA facilitating transcription of the sigE gene thereby leading finally to the enhancement of the transcription of rel in M. smegmatis and M. tuberculosis. Downregulation of ppk1 led to impaired survival of M. tuberculosis in macrophages. PolyP plays a central role in the stress response of mycobacteria

Understanding the role of the Sonic Hedgehog signaling pathway in cerebellar development and medulloblastoma genesis

Thesis (Ph.D.)--University of Washington, 2012Medulloblastoma is a developmental cancer of the cerebellum. It continues to be the most common pediatric brain cancer associated with dire survival and impaired quality of life. In order to develop therapeutic interventions, it is necessary to bridge the gaps in knowledge about cerebellar development and understand how aberrations in developmental pathways lead to cancer. The Sonic hedgehog pathway (Shh) plays a pivotal role in cerebellar development and mutations leading to hyperactive signaling cause medulloblastoma. Although the fundamentals of the pathway mechanics are known, and have led to the development of mouse models to study the human disease, there are critical questions that remain to be answered. Based on pathway signatures medulloblastomas are categorized into subgroups, Shh-driven being one such subgroup. However there is significant heterogeneity even among Shh-driven medulloblastomas that necessitates understanding key differences between the various mutations and differential regulation of downstream effectors. Through the characterization of a novel mouse model of medulloblastoma, SmoA2 and comparative analyses with the existing SmoA1 model, I have demonstrated salient molecular and cellular differences between two activating mutations in the same region of a single gene. While the SmoA1 mutation leads to medulloblastoma in adult mice, in addition to cancer, the SmoA2 mutation causes severe defects early in cerebellar development. The transcriptional profiles downstream of these two mutations and biological processes affected are distinct. An unexpected finding from the SmoA2 model is the preservation of normal cerebellar function despite a completely disrupted cytoarchitecture challenging the notion that stereotypical organization of the cerebellum is critical for its function. My second aim was to identify molecules that interact with the Shh pathway in development and disease. Toward this goal, I discovered a previously unknown expression pattern of MyoD in the proliferative phase of the developing cerebellum as well as in mouse medulloblastoma. MyoD, a myogenic differentiation factor has been known to be exclusive to the skeletal muscle lineage. I demonstrate MyoD functions as a novel haploinsufficient tumor suppressor in the context of medulloblastoma with potential clinical significance

A Platform for Rapid, Quantitative Assessment of Multiple Drug Combinations Simultaneously in Solid Tumors In Vivo.

While advances in high-throughput screening have resulted in increased ability to identify synergistic anti-cancer drug combinations, validation of drug synergy in the in vivo setting and prioritization of combinations for clinical development remain low-throughput and resource intensive. Furthermore, there is currently no viable method for prospectively assessing drug synergy directly in human patients in order to potentially tailor therapies. To address these issues we have employed the previously described CIVO platform and developed a quantitative approach for investigating multiple combination hypotheses simultaneously in single living tumors. This platform provides a rapid, quantitative and cost effective approach to compare and prioritize drug combinations based on evidence of synergistic tumor cell killing in the live tumor context. Using a gemcitabine resistant model of pancreatic cancer, we efficiently investigated nine rationally selected Abraxane-based combinations employing only 19 xenografted mice. Among the drugs tested, the BCL2/BCLxL inhibitor ABT-263 was identified as the one agent that synergized with Abraxane® to enhance acute induction of localized apoptosis in this model of human pancreatic cancer. Importantly, results obtained with CIVO accurately predicted the outcome of systemic dosing studies in the same model where superior tumor regression induced by the Abraxane/ABT-263 combination was observed compared to that induced by either single agent. This supports expanded use of CIVO as an in vivo platform for expedited in vivo drug combination validation and sets the stage for performing toxicity-sparing drug combination studies directly in cancer patients with solid malignancies

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer

Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer

Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development

CIVO screen for drugs that are synergistic with pancreatic cancer standard of care Abraxane^® in a Gemcitabine resistant xenograft model.

<p>CIVO screen for drugs that are synergistic with pancreatic cancer standard of care Abraxane^® in a Gemcitabine resistant xenograft model.</p