Project description:Acute Myeloid Leukemia (AML) which occurs after an antecedent myeloproliferative neoplasm (MPN) has a dismal clinical prognosis and is not curative outside of the rare subset of patients who undergo successful allogeneic stem cell transplantation. As such, there is a pressing need for new mechanistic insights into how MPNs transform into AML and to use these insights to credential novel therapeutic approaches. The most common somatic mutational event which occurs in transformation from MPN to AML is mutation in TP53. However, the impact of TP53 allelic state on the ability to potentiate leukemic transformation, as well as the pathways involved in this process, have largely remained unresolved. Here we report the development of genetically accurate models of Jak2/Tp53 mutant MPN which undergoes progressive leukemic transformation with chromosomal instability similar to that observed in the clinical context. These models result in a fulminant erythroleukemia phenotype. We identify that leukemic transformation requires homozygous inactivation of TP53, and does not occur with heterozygous loss of Tp53. We further identify that the megakaryocyte erythroid progenitor (MEP) population is expanded prior to and after leukemic transformation, is characterized by progressive genomic instability (compared to other stem/progenitor compartments), and is capable of propagating the disease in vivo. Thus, the leukemia-initiating population is contained in the MEP compartment. Using gene-expression profiling we demonstrate that the BMP2/SMAD pathway (which is involved in self-renewal and DNA damage repair) is aberrantly activated in the leukemic phase of the disease. Importantly, attenuation of Bmp2 results in decreased self-renewal of leukemic cells in vitro and increased survival of leukemic mice in vivo, thus credentialing a biologic role for this pathway in leukemic transformation. Finally, given the loss of Tp53 function and associated disruption of DNA repair pathways, we hypothesized and subsequently identified that leukemic transformation is characterized by increased DNA damage. Using a synthetic-lethality strategy of targeting remaining DNA-repair pathways, using small-molecule inhibitors, in order to provoke biologically intolerable DNA damage and mitotic catastrophe, we demonstrate that Jak2/Tp53 mutant is highly sensitive to combined inhibition of WEE1 and PARP. This combination results in prolonged survival of mice and attenuates the leukemic phenotype. Collectively, these observations yield new mechanistic insights into the process of leukemic transformation resulting from TP53 alterations, and offer new, clinically-translatable, therapeutic options.

Project description:Epigenetically silenced Ink4a-Arf locus is activated by loss of H3K27me3 in cellular senescence, where secreted factor expression is also involved. Here we analyzed epigenome and transcriptome alteration during Ras-induced senescence using mouse embryonic fibroblast (MEF). Seventeen genes with H3K27me3 loss and H3K4me3 gain showed marked upregulation, including p16Ink4a and Bmp2, a secreted factor for BMP/SMAD signal. Smad6, specific BMP/SMAD pathway inhibitor, was identified as the only one gene showing de novo H3K27 trimethylation with H3K4me3, resulting in strong repression. Ras-activated cells senesced with SMAD1/5/8 phosphorylation, and they escaped from senescence with decreased SMAD1/5/8 phosphorylation when introducing Smad6 or knocking-down Bmp2. Mouse embryonic fibroblasts were established from 13.5 embryonic day embryos of C57/B6. After cells were passaged twice (MEFp2), cells were infected with retroviruses for 48 hours. Then cells were exposed to 4 M-NM-<g/mL peuromycin for selection during days 0-3, and were passed on days 3, 7, and 10. Retroviral vectors for Ras was constructed by cloning cDNAs for wild type HRAS (RasG12) and mutated HRAS (RasV12) by reverse-transcription PCR products from HMEC and SK-BR3 cell RNA, respectively, with N-terminal FLAG tag into pMX vector that contains puromycin resistance gene. Mock pMX vector (mock), and vectors containing RasG12 and oncogenic RasV12 were transfected into plat-E packaging cells using FuGENE 6 Transfection Reagent (Roche, Germany) to prepare retroviruses. Smad6 cDNA with N-termainal 6x Myc tag was also cloned into pMX vector. To knock down Bmp2, double strand oligonucleotide DNA to express small hairpin RNA against Bmp2 (shBmp2) was cloned into RNAi-Ready pSIREN-RetroQ Vector (Clontech, CA). Viral packaging for Smad6 and shBmp2 retrovirus vectors was also done using plat-E cells. For genome-wide transcription analysis, GeneChip Mouse Genome 430 2.0 Array (Affimetrix) was used. For global normalization, the average signal in an array was made equal to 100. Chromatin immunoprecipitation (ChIP)-sequencing was performed. MEFp2 cells and cells with mock, RasG12 or RasV12 infection at day 10 were cross-linked with 1% formaldehyde for 10 min at room temperature and were prepared for ChIP. ChIP using anti-H3K4me3 (ab8580, abcam, rabbit polyclonal) or H3K27me3 (07-142, Upstate, rabbit polyclonal) antibody was performed as described previously. Sample preparation for ChIP-sequencing was performed according to the manufacturer's instructions (Ilumina), and sequencing was performed using Solexa Giga sequencer.

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The BMP2/SMAD pathway is a key regulator of TP53-altered erytholeukemia

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