Pier Paolo Pandolfi - US grants

1R01 DK115536-01 revised Inherited bone marrow failure disorders (BMFDs) represent a distinct category of hematopoietic disorders that are driven by genetic mutation. Within these disorders, a distinct set of genes has already been identified that contribute to a sub-set of BMFDs known as ribosomopathies. Ribosomopathies harbor mutation to genes playing a critical role in ribosomal processing and ribosome biogenesis. We previously established the DKC1 gene as a ribosomopathy gene through its ability to regulate proper translation as a result of its function to convert uridine residues on ribosomal RNAs (rRNAs) to pseudouridine. Exciting, preliminary data from our lab now demonstrates that another post-transcriptional modification of rRNAs, 2?-O-methylation (2?-O-Me), also contribute to proper regulation of ribosome function. Our data reveal that specific C/D-box small nucleolar RNAs (snoRNAs) controls proper IRES-dependent translation of major cell cycle and apoptosis genes. In vivo, disruption of 2?-O-Me in adult mouse hematopoietic stem cell (HSC) compartment results in features characteristic of ribosomopathies such as defects in stem cell maintenance due to exit from quiescence, apoptosis, and myelodysplastic bone marrow failure. Importantly, we identify novel germ line mutations dyskeratosis congenita (DC) patients related to function of C/D box snoRNAs. Thus, our preliminary findings provide direct genetic evidence for the critical role ribosome specialization through rRNA 2?-O-Me, as well as in the pathogenesis of multiple disease states through aberrant HSC and ribosome function. In order to better understand the role and function 2?-O-Me, we propose to (1) study the molecular and cellular pathways that are impacted by 2?-O-Me and which contribute to the process of bone marrow failure, (2) determine the in vivo significance of normal ribosome function for hematopoiesis, and of novel DC mutations identified through development of genetically engineered mouse models, and (3) evaluate the extent to which genes involved in the biochemical complexes that catalyzes 2?-O-Me may be altered in BMFDs, and to determine if additional novel pathogenic mutations targeting the process of rRNA methylation, including snoRNAs, exist in BMFDs. Together these data will further facilitate our understanding of ribosomopathies, and help uncover how essential regulation of the ribosome, through rRNA modification, contributes to normal and aberrant hematopoiesis.

Summary: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most commonly altered tumor suppressor genes in human cancers. It has been shown that tumor susceptibility is highly sensitive to cellular PTEN levels, thereby highlighting the importance of molecular mechanisms for PTEN regulation. Recent research has uncovered a novel mechanism for post-transcriptional regulation of PTEN via a network of microRNAs (miRNAs) and competing endogenous RNAs (ceRNAs). Currently known ceRNAs that regulate PTEN are limited to protein-coding mRNAs and a quantitative framework for modeling PTEN regulation by ceRNAs is lacking. The project proposes to develop computational and experimental approaches for the discovery and analysis of both non-coding and coding ceRNAs of PTEN. The proposed research will lead to the identification of multiple ceRNAs of PTEN and focus on their role in controlling cellular PTEN levels and their impact on tumor susceptibility. The long-term objective is to develop novel therapeutic approaches for cancer based on elevating cellular PTEN levels using ceRNA-based regulation. The specific aims are to: 1) Develop a quantitative model for kinetics and regulation of PTEN by ceRNAs; 2) Develop quantitative models of post-transcriptional regulation of PTEN by ceRNA networks; and 3) Develop protocols for controlling PTEN using ceRNAs and determine the impact on tumor susceptibility. This project involves the innovative integration of approaches from different disciplines and tools such as single-cell assays, stochastic modeling, machine learning, and bioinformatics to analyze regulation of PTEN via ceRNAs. The development of quantitative models and tools for analysis of ceRNA-based regulation, will significantly impact current and future research aimed at understanding its role in diverse cellular processes, thereby significantly impacting the field beyond PTEN function. The project will strengthen collaboration between different departments at UMass Boston and DF/HCC, and will provide excellent interdisciplinary training and mentorship for students and researchers involved. Project Co-Leads, Zarringhalam and Kulkarni, are mentoring several undergraduate students at UMass Boston who are currently working on the project. Students have written Honors theses, given talks based on their research at undergraduate conferences, and are co-authors on multiple presentations at prestigious international conferences. Providing ample opportunity for research experiences for students from underrepresented backgrounds is a top priority of the project, and the project Co-Leads will work diligently with the U54 Research Education Core to facilitate these experiences. Besides the positives arising from interdisciplinary training for students in cutting-edge research, the wide dissemination of the results will also contribute positively to the UMass Boston-DF/HCC U54 Partnership, attract external funding and lead to the development of a strong cancer research program at UMass Boston.

? DESCRIPTION (provided by applicant): Relapsed disease following conventional therapy remains one of the central problems in the treatment of leukemia. Leukemia initiating cells (LIC), possess stem cell properties such as self-renewal and pluripotency enabling them to mediate disease initiation and maintenance. Targeting the LIC compartment is therefore at the core of novel therapeutic approaches to leukemia. We have demonstrated that the Promyelocytic Leukemia gene (PML) is critical to HSC and CML LIC maintenance. Notably, pharmacologic ablation of PML using arsenic trioxide (ATO) combined with cytotoxic therapy resulted in the exhaustion of LICs and eradication of disease in a BCR-ABL CML murine model. Our preclinical findings led to a phase I trial at our institution combining TKI and ATO in CML patients with evidence of persistent disease, as well as a multi-center, randomized trial in China comparing TKI and TKI plus a novel oral arsenic in CML. Encouraging interim results of our own trial prompted the addition of a new site as well as collaboration with our colleagues in China. Our ongoing studies of PML's role in HSC and LIC maintenance has uncovered evidence of a novel non-cell- autonomous role for PML in HSCs and in CML and AML LICs through its expression in mesenchymal stem cells suggesting that PML targeting strategies could be dually effective against the leukemic niche and LIC proper. Furthermore, we discovered a cell-autonomous, metabolic dimension to PML-orchestrated HSC maintenance via the nutrient sensor, peroxisome proliferator activating receptor ? (PPAR ?) and fatty acid oxidation (FAO) which promote HSC asymmetric division. In AML, we have intriguing evidence that abrogating PPAR signaling with a novel PPAR? inhibitor markedly inhibits colony initiating capacity. In addition, our preliminary data indicates that the combination of ATO and PPAR inhibition results in synergistic cytotoxicity in part through increased ROS production. Given the ROS-low status of the AML LIC compartment, we postulate that this combination could efficiently target the LIC compartment in specific subtypes of AML, such as IDH mutant leukemia, which is known to have limited reducing capacity. To translate the potential of targeting PML pathways, we propose to conduct the following Specific Aims: (1) to define the subtypes of AML that rely on Pml expression in MSCs for LIC maintenance and dissect the MSC-derived factors that mediate LIC maintenance in CML and AML (2) to study the role of the PML/PPAR/FAO pathway in AML LICs; (3) to assess the effectiveness of combinatorial treatments targeting PML pathways, against AML LICs in preclinical murine models, including IDH2-mutant models, with evaluation of PML targeting strategies in combination with IDH2-mutant deinduction and (4) to develop a clinical trial of PPAR? inhibition alone or in combination with ATO for the treatment of relapsed, refractory AML. The studies proposed will significantly impact the treatment of CML and AML patients by targeting the reservoir of leukemic cells that mediates disease persistence and relapse.

? DESCRIPTION (provided by applicant): Acute promyelocytic leukemia (APL) is associated with reciprocal translocations that always involve the RAR? locus on chromosome 17, which variably translocates and fuses to the PML, PLZF, NPM1 (for brevity referred to as X genes/proteins). We originally hypothesized that X-RAR? and RAR?-X would act to interfere with both the transcriptional function of RAR??and the biological function of X proteins, and tat X proteins would thus play a key role in leukemogenesis and oncogenesis. Fundamental to this proposal, we also hypothesized, that the function of the X proteins of APL may be perturbed or lost in malignancies other than APL, and that the X proteins could share common activities and biological roles, as APL is ultimately the invariable outcome of their perturbed functions. On this basis, we proposed to study the various X genes comparatively and systematically in their physiologic and developmental roles as well as in the pathogenesis of human cancer through a direct genetic approach in the mouse as well as in human primary cancers of various histology. With this approach we have already accrued a wealth of information through the analysis of complete knock-out (KO) mouse mutant strains and primary cells/tissues from these mutants and human cancers. This analysis led to fundamental groundbreaking discoveries and strongly supported the hypothesis that X genes are not only critical cancer genes, but in fact tumor suppressor genes (TSGs) whose function is broadly perturbed in human cancer, well beyond the pathogenesis of APL. These discoveries have established me as an outstanding investigator in the field of TSG biology and regulation, and have been recognized at multiple levels with more than 20 national and international awards, numerous invited Keynote lectures and other accolades. We now propose to continue to study these critical genes as paradigms for tumor suppression, through the development of a second generation of models and tools in order to explore how they function in leukemia and other cancers, and, importantly, to develop and test new cancer therapies. To this end, we will adopt three main strategies: (1) To generate a second generation of conditional-tissue specific, cancer relevant, mutants for each of the genes in question to address cell specific and non-adaptive functions of these TSGs; (2) To characterize new regulatory mechanisms of these TSGs, including the non-coding RNAs dimension, to develop a set of criteria and novel concepts that defines how TSG function can be perturbed towards cancer initiation and progression; and (3) To develop and test novel therapeutic approaches that target TSG deficiency, targeting key pathways as well as signaling and regulatory nodes identified throughout our studies.