Unique Transcriptional Profiles Underlie Osteosarcomagenesis Driven by Different p53 Mutants

UNLABELLED: Missense mutations in the DNA binding domain of p53 are characterized as structural or contact mutations based on their effect on the conformation of the protein. These mutations show gain-of-function (GOF) activities, such as promoting increased metastatic incidence compared with p53 loss, often mediated by the interaction of mutant p53 with a set of transcription factors. These interactions are largely context specific. To understand the mechanisms by which p53 DNA binding domain mutations drive osteosarcoma progression, we created mouse models, in which either the p53 structural mutant p53R172H or the contact mutant p53R245W are expressed specifically in osteoblasts, yielding osteosarcoma tumor development. Survival significantly decreased and metastatic incidence increased in mice expressing p53 mutants compared with p53-null mice, suggesting GOF. RNA sequencing of primary osteosarcomas revealed vastly different gene expression profiles between tumors expressing the missense mutants and p53-null tumors. Further, p53R172H and p53R245W each regulated unique transcriptomes and pathways through interactions with a distinct repertoire of transcription factors. Validation assays showed that p53R245W, but not p53R172H, interacts with KLF15 to drive migration and invasion in osteosarcoma cell lines and promotes metastasis in allogeneic transplantation models. In addition, analyses of p53R248W chromatin immunoprecipitation peaks showed enrichment of KLF15 motifs in human osteoblasts. Taken together, these data identify unique mechanisms of action of the structural and contact mutants of p53. SIGNIFICANCE: The p53 DNA binding domain contact mutant p53R245W, but not the structural mutant p53R172H, interacts with KLF15 to drive metastasis in somatic osteosarcoma, providing a potential vulnerability in tumors expressing p53R245W mutation

Inhibition of translation initiation factor eIF4a inactivates heat shock factor 1 (HSF1) and exerts anti-leukemia activity in AML

Eukaryotic initiation factor 4A (eIF4A), the enzymatic core of the eIF4F complex essential for translation initiation, plays a key role in the oncogenic reprogramming of protein synthesis, and thus is a putative therapeutic target in cancer. As important component of its anticancer activity, inhibition of translation initiation can alleviate oncogenic activation of HSF1, a stress-inducible transcription factor that enables cancer cell growth and survival. Here, we show that primary acute myeloid leukemia (AML) cells exhibit the highest transcript levels of eIF4A1 compared to other cancer types. eIF4A inhibition by the potent and specific compound rohinitib (RHT) inactivated HSF1 in these cells, and exerted pronounced in vitro and in vivo anti-leukemia effects against progenitor and leukemia-initiating cells, especially those with FLT3-internal tandem duplication (ITD). In addition to its own anti-leukemic activity, genetic knockdown of HSF1 also sensitized FLT3-mutant AML cells to clinical FLT3 inhibitors, and this synergy was conserved in FLT3 double-mutant cells carrying both ITD and tyrosine kinase domain mutations. Consistently, the combination of RHT and FLT3 inhibitors was highly synergistic in primary FLT3-mutated AML cells. Our results provide a novel therapeutic rationale for co-targeting eIF4A and FLT3 to address the clinical challenge of treating FLT3-mutant AML.R01 CA175744 - NCI NIH HHS; R35 GM118173 - NIGMS NIH HHS; P30 CA016672 - NCI NIH HHSPublished versionSupporting documentationAccepted manuscrip

UNIQUE TRANSCRIPTIONAL PROFILES UNDERLIE OSTEOSARCOMAGENESIS DRIVEN BY DIFFERENT p53 MUTANTS

Missense mutations in the DNA binding domain of the Trp53 gene are characterized as structural (p53R172H) or contact (p53R245W) mutations based on their effect on the conformation of the protein. These mutations show gain-of-function activities such as increased metastatic incidence as compared to p53 loss, often mediated by their interaction with a repertoire of transcription factors. These interactions are largely context specific. In order to understand the mechanisms by which these mutations drive osteosarcoma progression, we created a mouse model, wherein either the p53 structural mutant p53R172H, or the contact mutant, p53R245W, are expressed specifically in osteoblasts, yielding osteosarcoma tumor development. We observed a significant decrease in survival and increased metastatic incidence in mice expressing p53 mutants as compared to p53-null mice, suggesting gain of function. RNA-sequencing of primary osteosarcomas revealed that tumors expressing these missense mutants had vastly different gene expression profiles as compared to p53-null tumors. Further, p53R172H and p53R245W regulated unique transcriptomes, that affected distinct pathways, through interactions with a unique repertoire of transcription factors. Validation assays showed that p53R245W, but not p53R172H, interacts with KLF15 to drive migration and invasion in osteosarcoma cell lines, and metastases in allo-transplantation models. Additionally, analyses of p53R248W ChIP peaks showed enrichment of KLF15 motifs in human osteoblasts. Taken together, these data suggest that the structural and contact mutants of p53 have unique mechanisms of action

The Effects of Pulsed Alcohol Consumption on Mouse Mammary Tumorigenesis

Alcohol has been implicated as the only dietary factor that increases the risk of breast cancer in women. It is not known whether the effects of alcohol exposure are the same throughout the lifetime, or if alcohol exerts a greater effect at times of rapid remodeling of breast tissue, such as puberty or post-lactational involution, compared to a resting adult state. The purpose of this study is to define the effects of short term pulsing with ethanol on subsequent tumor development and metastasis in resting, nonparous mice. These resting mice serve as age-matched controls for a larger study comparing ethanol’s effects on parous mice when administered during post-lactational involution. For this project, transgenic FVB-MMTV- HER2/neu mice, which overexpress the epidermal growth factor receptor type 2 in the mammary gland, are used to model the etiology of HER2-overexpressing human breast cancers. Nonparous female mice, aged 70-85 days, were administered 0, 0.5%, 1%, or 2% alcohol in liquid diets for a 2 week period, and then evaluated for tumorigenesis over the following 9 months. No significant difference is observed between control or alcohol-fed mice in terms of tumor latency, multiplicity, burden, or growth rate. Alcohol feeding likewise has no effect on frequency of lung metastases. However, nonparous mice show an increased latency compared to parous mice, confirming that parity accelerates tumorigenesis. These results show that alcohol consumption for short time periods has no effect on HER2-mediated mammary tumorigenesis in the nonparous mouse

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs

<div><p>BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.</p></div

Apoptosis analysis of AML cells with p53 knockdown.

<p>MOLM-13, MV4;11 and OCI-AML3 cell lines transfected with a lentivirus carrying scrambled shRNA (ShC) or a shRNA specific for p53 (ShP53) were treated with (A) Nutlin-3a (0, 0.5, 1.0, 2.5, 5, or 10 μM), Ara-C (0, 0.5, 1.0, 2.5, 5, 10, or 20 μM), KPT-330 (0, 50, 100, 250, 500, 1000 nM in MOLM-13 and MV4;11 cells. 0, 50, 100, 250, 500, 1000, 2500, 5000, and 10,000 nM in OCI-AML3 lines), or (B) ABT-199 (0, 50, 100, 250, 500, 1000 nM in MOLM-13 and MV4;11 cells. 0, 50, 100, 250, 500, 1000, 2500, 5000, and 10,000 nM in OCI-AML3 lines) for 48 hours. The Annexin V-positive cell fractions and Annexin V- and propidium iodide-negative live cells were counted using flow cytometry. All experiments were performed in duplicate. The y axes of left graphs show the extent of drug-specific apoptosis, calculated as described in the method section.</p