Search

Supplementary Data from The MDM2-A Splice Variant of MDM2 Alters Transformation In vitro and the Tumor Spectrum in Both Arf- and p53-Null Models of Tumorigenesis

Strong nuclear staining of mutant TP53-R337H protein in normal tissue of carriers who developed ACC.

Mouse Cohorts and Associated Pathology

Angiosarcoma is an aggressive vascular sarcoma with an extremely poor prognosis. Because of the relative rarity of this disease, its molecular drivers and optimal treatment strategies are obscure. DICER1 is an RNase III endoribonuclease central to miRNA biogenesis, and germline DICER1 mutations result in a cancer predisposition syndrome, associated with an increased risk of many tumor types. Here, we show that biallelic Dicer1 deletion with aP2-Cre drives aggressive and metastatic angiosarcoma independent of other genetically engineered oncogenes or tumor suppressor loss. Angiosarcomas in aP2-Cre;Dicer1Flox/- mice histologically and genetically resemble human angiosarcoma. miR-23 target genes, including the oncogenes Ccnd1 as well as Adam19, Plau, and Wsb1 that promote invasiveness and metastasis, were enriched in mouse and human angiosarcoma. These studies illustrate that Dicer1 can function as a traditional loss-of-function tumor suppressor gene, and they provide a fully penetrant animal model for the study of angiosarcoma development and metastasis. Cancer Res; 77(22); 6109–18. ©2017 AACR.

This file contains four supplementary tables and four supplementary figures pertinent to the submitted manuscript as follows: Table S1 lists the Enrichr analysis of miRNA targets in angiosarcoma. Table S2 lists the antibodies used for immunohistochemistry. Table S3 lists the SYBR primers and Taqman probes used for qRT-PCR. Table S4 lists the antibodies used for immunoblots. Figure S1 shows Dicer1 deletion in adipose tissue. Figure S2 displays angiosarcomas in the lungs of aP2-Cre;Dicer1Flox/- mice. Figure S3 compares mouse and human angiosarcomas. Figure S4 illustrates the interaction of the miR~23~27~24 cluster with gene targets in angiosarcoma.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li–Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up.Significance:A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

Supplemental Materials and Methods and Supplementary Figure Legends

Supplementary Figures 1-7 from p18Ink4c and p53 Act as Tumor Suppressors in Cyclin D1–Driven Primitive Neuroectodermal Tumor

Supplementary Figure 2 from Genetic Alterations in Mouse Medulloblastomas and Generation of Tumors De novo from Primary Cerebellar Granule Neuron Precursors

Mice lacking p53 and one or two alleles of the cyclin D–dependent kinase inhibitor p18Ink4c are prone to medulloblastoma development. The tumor frequency is increased by exposing postnatal animals to ionizing radiation at a time when their cerebella are developing. In irradiated mice engineered to express a floxed p53 allele and a Nestin-Cre transgene, tumor development can be restricted to the brain. Analysis of these animals indicated that inactivation of one or both Ink4c alleles did not affect the time of medulloblastoma onset but increased tumor invasiveness. All such tumors exhibited complete loss of function of the Patched 1 (Ptc1) gene encoding the receptor for sonic hedgehog, and many exhibited other recurrent genetic alterations, including trisomy of chromosome 6, amplification of N-Myc, modest increases in copy number of the Ccnd1 gene encoding cyclin D1, and other complex chromosomal rearrangements. In contrast, medulloblastomas arising in Ptc1+/− mice lacking one or both Ink4c alleles retained p53 function and exhibited only limited genomic instability. Nonetheless, complete inactivation of the wild-type Ptc1 allele was a universal event, and trisomy of chromosome 6 was again frequent. The enforced expression of N-Myc or cyclin D1 in primary cerebellar granule neuron precursors isolated from Ink4c−/−, p53−/− mice enabled the cells to initiate medulloblastomas when injected back into the brains of immunocompromised recipient animals. These “engineered” tumors exhibited gene expression profiles indistinguishable from those of medulloblastomas that arose spontaneously. These results underscore the functional interplay between a network of specific genes that recurrently contribute to medulloblastoma formation. [Cancer Res 2007;67(6):2676–84]

Supplementary Figure 1 from Genetic Alterations in Mouse Medulloblastomas and Generation of Tumors De novo from Primary Cerebellar Granule Neuron Precursors

XLSX file - 146K, Table S1- Data on the genes that differ in expression between primary tumors Th- MYCN, Caspase-8 wild type levels with no BMM, and primary tumors Th-MYCN, deficient caspase-8 with BMM (p

PDF file - 709K, Figure S1. Correlation of relative mean fold changes in expression of genes and microRNAs of the mouse tumor groups by expression- array and by quantitative RT-PCR. Figure S2. Levels of caspase-8 protein in primary tumors and BM secondary tumors. Figure S3. Primary tumors from wild-type or caspase-8 deficient (fl/ko, fl/fl or wt/ko caspase-8) mice had neuroblastoma morphology and stained positive for neuronal markers typical for neuroblastoma. Figure S4. Age at maximum tumor burden of neuroblastoma primary tumors in the Th-MYCN mouse groups. Figure S5. Measuring apoptosis levels by immunostaining for cleaved caspase-3. Figure S6. MYCN expression in the primary Th-MYCN, Th-Cre neuroblastoma tumors. Figure S7. Secondary tumors observed in both the Th-MYCN Caspase-8 wt/wt and the Th-MYCN deficient caspase-8 mouse models.

XLSX file - 77K, Table S3- Data on the microRNAs that differ in expression between primary tumors Th- MYCN, Caspase-8 wild type levels with no BMM, and primary tumors Th-MYCN, deficient caspase-8 with BMM (p

Supplementary Figure Legends 1-2 from Genetic Alterations in Mouse Medulloblastomas and Generation of Tumors De novo from Primary Cerebellar Granule Neuron Precursors

Supplementary Table 1 from Genetic Alterations in Mouse Medulloblastomas and Generation of Tumors De novo from Primary Cerebellar Granule Neuron Precursors

Multiple myeloma (MM) is an incurable plasma cell malignancy. The recent successes of the proteasome inhibitor bortezomib in MM therapy have prompted investigations of its efficacy in combination with other anticancer agents. Polyamines play important roles in regulating tumor cell proliferation and angiogenesis and represent an important therapeutic target. CGC-11093 is a novel polyamine analogue that has completed a phase I clinical trial for the treatment of cancer. Here, we report that CGC-11093 selectively augments the in vitro and in vivo antimyeloma activity of bortezomib. Specifically, the combination of CGC-11093 and bortezomib compromised MM viability and clonogenic survival, and increased drug-induced apoptosis over that achieved by either single agent. Xenografts of MM tumors treated with this combination had marked increases in phospho-c-Jun-NH2-kinase (JNK)-positive cells and apoptosis, and corresponding reductions in tumor burden, tumor vasculature, and the expression of proliferating cell nuclear antigen and the proangiogenic cytokine vascular endothelial growth factor. Furthermore, inhibition of JNK with a pharmacologic inhibitor or by selective knockdown blunted the efficacy of CGC-11093 and bortezomib. Therefore, CGC-11093 enhances the anticancer activity of bortezomib by augmenting JNK-mediated apoptosis and blocking angiogenesis. These findings support the study of the use of the combination of bortezomib and CGC-11093 in MM patients that fail to respond to frontline therapy. [Cancer Res 2008;68(12):4783–90]

Supplementary Methods and Figure 1 from The Novel Polyamine Analogue CGC-11093 Enhances the Antimyeloma Activity of Bortezomib

Neuroblastoma, the most common extracranial pediatric solid tumor, is responsible for 15% of all childhood cancer deaths. Patients frequently present at diagnosis with metastatic disease, particularly to the bone marrow. Advances in therapy and understanding of the metastatic process have been limited due, in part, to the lack of animal models harboring bone marrow disease. The widely used transgenic model, the Th-MYCN mouse, exhibits limited metastasis to this site. Here, we establish the first genetic immunocompetent mouse model for metastatic neuroblastoma with enhanced secondary tumors in the bone marrow. This model recapitulates 2 frequent alterations in metastatic neuroblasoma, overexpression of MYCN and loss of caspase-8 expression. Mouse caspase-8 gene was deleted in neural crest lineage cells by crossing a Th-Cre transgenic mouse with a caspase-8 conditional knockout mouse. This mouse was then crossed with the neuroblastoma prone Th-MYCN mouse. Although overexpression of MYCN by itself rarely caused bone marrow metastasis, combining MYCN overexpression and caspase-8 deletion significantly enhanced bone marrow metastasis (37% incidence). Microarray expression studies of the primary tumors mRNAs and microRNAs revealed extracellular matrix structural changes, increased expression of genes involved in epithelial to mesenchymal transition, inflammation, and downregulation of miR-7a and miR-29b. These molecular changes have been shown to be associated with tumor progression and activation of the cytokine TGF-β pathway in various tumor models. Cytokine TGF-β can preferentially promote single cell motility and blood-borne metastasis and therefore activation of this pathway may explain the enhanced bone marrow metastasis observed in this animal model. Cancer Res; 73(13); 4086–97. ©2013 AACR.

XLSX file - 175K, Table S2- Data on the genes that differ in expression between primary tumors Th- MYCN, Caspase-8 wild type with no BMM, and primary tumors Th-MYCN, deficient caspase-8 without BMM (p

Background Pediatric high-grade glioma (pHGG) is largely incurable and accounts for most brain tumor-related deaths in children. Radiation is a standard therapy, yet the benefit from this treatment modality is transient, and most children succumb to disease within 2 years. Recent large-scale genomic studies suggest that pHGG has alterations in DNA damage response (DDR) pathways that induce resistance to DNA damaging agents. The aim of this study was to evaluate the therapeutic potential and molecular consequences of combining radiation with selective DDR inhibition in pHGG. Methods We conducted an unbiased screen in pHGG cells that combined radiation with clinical candidates targeting the DDR and identified the ATM inhibitor AZD1390. Subsequently, we profiled AZD1390 + radiation in an extensive panel of early passage pHGG cell lines, mechanistically characterized response to the combination in vitro in sensitive and resistant cells and evaluated the combination in vivo using TP53 wild-type and TP53 mutant orthotopic xenografts. Results AZD1390 significantly potentiated radiation across molecular subgroups of pHGG by increasing mutagenic nonhomologous end joining and augmenting genomic instability. In contrast to previous reports, ATM inhibition significantly improved the efficacy of radiation in both TP53 wild-type and TP53 mutant isogenic cell lines and distinct orthotopic xenograft models. Furthermore, we identified a novel mechanism of resistance to AZD1390 + radiation that was marked by an attenuated ATM pathway response which dampened sensitivity to ATM inhibition and induced synthetic lethality with ATR inhibition. Conclusions Our study supports the clinical evaluation of AZD1390 in combination with radiation in pediatric patients with HGG.

Angiosarcomas are aggressive vascular sarcomas that arise from endothelial cells and have an extremely poor prognosis. Because of the rarity of angiosarcomas, knowledge of molecular drivers and optimized treatment strategies is lacking, highlighting the need for in vivo models to study the disease. Previously, we generated genetically engineered mouse models of angiosarcoma driven by aP2-Cre-mediated biallelic loss of Dicer1 or conditional activation of Kras

PTEN promoter hypermethylation is nearly universal and PTEN copy number loss occurs in ~25% of fusion-negative rhabdomyosarcoma (FN-RMS). Here we show Pten deletion in a mouse model of FN-RMS results in less differentiated tumors more closely resembling human embryonal RMS. PTEN loss activated the PI3K pathway but did not increase mTOR activity. In wild-type tumors, PTEN was expressed in the nucleus suggesting loss of nuclear PTEN functions could account for these phenotypes. Pten deleted tumors had increased expression of transcription factors important in neural and skeletal muscle development including Dbx1 and Pax7. Pax7 deletion completely rescued the effects of Pten loss. Strikingly, these Pten;Pax7 deleted tumors were no longer FN-RMS but displayed smooth muscle differentiation similar to leiomyosarcoma. These data highlight how Pten loss in FN-RMS is connected to a PAX7 lineage-specific transcriptional output that creates a dependency or synthetic essentiality on the transcription factor PAX7 to maintain tumor identity., PTEN copy number loss is found in 25% of fusion-negative rhabdomyosarcomas (FN-RMS). Here, the authors use a Hedgehog-driven FN-RMS mouse model to show that PTEN loss drives the expression of core transcription factor PAX7 and its transcriptional axis, which determines FN-RMS tumour identity.

Aspartame, an artificial sweetener commonly used as a sugar substitute, is currently authorized for use in more than 100 countries. Hundreds of studies, conducted in various countries dating back to the 1970s, have shown that aspartame is safe at real-world exposure levels. Furthermore, multiple human epidemiology studies have provided no indication that consumption of aspartame induces cancer. Given the continued controversy surrounding the Ramazzini Institute's (RI) studies suggesting that aspartame is a carcinogenic hazard in rodents and evaluation by the International Agency for Research on Cancer, this report aims to provide the perspective of experienced pathologists on publicly available pathology data regarding purported proliferative lesions in liver, lung, lymphoid organs, and mammary gland as well as their implications for human risk assessment as reported for three lifetime rodent carcinogenicity bioassays of aspartame conducted at the RI. In the authors' view, flaws in the design, methodology and reporting of the RI aspartame studies limit the utility of the data sets as evidence that this agent represents a carcinogenic hazard. Therefore, all three RI studies, and particularly the accuracy of their pathology diagnoses and interpretations, should be rigorously reviewed by qualified and experienced veterinary toxicologic pathologists in assessing aspartame's carcinogenic risk.

Histiocytic sarcoma is a tumor of the hematopoietic system considered to be derived from macrophages. Although rare in humans, it occurs frequently in mice. Histiocytic sarcoma can be a difficult tumor to diagnose due to its diverse cellular morphologies, growth patterns, and organ distributions. The varying morphology of histiocytic sarcomas makes it easy to confuse them with other types of neoplasia, including hepatic hemangiosarcoma, uterine schwannoma, leiomyosarcoma, uterine stromal cell tumor, intramedullary osteosarcoma, and myeloid leukemia. As such, immunohistochemistry (IHC) is often needed to differentiate histiocytic sarcomas from other common tumors in mice that they can morphologically mimic. The goal of this article is to present a broader perspective of the diverse cellular morphologies, growth patterns, organ distributions, and IHC labeling of histiocytic sarcomas encountered by the authors. This article describes these features in a set of 62 mouse histiocytic sarcomas, including the IHC characterization of the tumors using a panel of markers for the macrophage antigens F4/80, IBA1, MAC2, CD163, CD68, and lysozyme, and describes differentiating features of histiocytic sarcomas from other morphologically similar tumors. The genetic changes underlying the pathogenesis of histiocytic sarcoma in humans are beginning to be elucidated, but this is difficult due to its rarity. The higher prevalence of this tumor in mice provides opportunities to investigate mechanisms of its development and to test potential treatments.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li–Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up. Significance: A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

The NOD.Cg- Prkdcscid Il2rgtm1Wjl/SzJ strain (NOD scid gamma, NSG) is a severely immunodeficient inbred laboratory mouse used for preclinical studies because it is amenable to engraftment with human cells. Combining scid and Il2rgnull mutations results in severe immunodeficiency by impairing the maturation, survival, and functionality of interleukin 2–dependent immune cells, including T, B, and natural killer lymphocytes. While NSG mice are reportedly resistant to developing spontaneous lymphomas/leukemias, there are reports of hematopoietic cancers developing. In this study, we characterized the immunophenotype of spontaneous lymphoma/leukemia in 12 NSG mice (20 to 38 weeks old). The mice had a combination of grossly enlarged thymus, spleen, or lymph nodes and variable histologic involvement of the bone marrow and other tissues. All 12 lymphomas were diffusely CD3, TDT, and CD4 positive, and 11 of 12 were also positive for CD8, which together was consistent with precursor T-cell lymphoblastic lymphoma/leukemia (pre-T-LBL). A subset of NSG tissues from all mice and neoplastic lymphocytes from 8 of 12 cases had strong immunoreactivity for retroviral p30 core protein, suggesting an association with a viral infection. These data highlight that NSG mice may develop T-cell lymphoma at low frequency, necessitating the recognition of this spontaneously arising disease when interpreting studies.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite aggressive treatment clinical outcomes for RMS have not improved for three decades, emphasizing the need to uncover the molecular underpinnings of the disease. RMS has been presumed to originate from derailed muscle progenitors based on the histologic appearance and gene expression pattern of the tumors resembling embryonic developing skeletal muscle. However, an origin restricted to skeletal muscle does not explain RMS occurring in tissues devoid of skeletal muscle such as the prostate, bladder, and salivary gland. Previously, we described that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of a presumed adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice gives rise to aggressive skeletal muscle tumors. These tumors display the histologic and molecular characteristics of human embryonal fusion-negative RMS (FN-RMS). This model suggested a potential non-myogenic origin of FN-RMS and an avenue to explain FN-RMS development in anatomic sites devoid of skeletal muscle. Lineage tracing showed that RMS can originate from cell reprogramming and transdifferentiation of endothelial progenitor cells. Hedgehog pathway activation in committed endothelial progenitors results in Tbx1 expression and subsequent Myod1 expression driving a partially myogenic program characteristic of FN-RMS. Our work identifies reprogramming cell fate as a mechanism of transformation in pediatric sarcoma and illustrates that it is dangerous to assume the cell of origin from the characteristics of the tumor cell. The cell-reprogramming mechanism that shifts endothelial progenitors to muscle-like cells provides a unique system to define the core regulatory circuitry controlling RMS cell fate and to determine in vivo if targeting this network is a therapeutic vulnerability. Genomic profiling of human FN-RMS failed to uncover a unique mutation that drives oncogenesis. However, the PTEN cis-regulatory region is hypermethylated in more than 90% of all human FN-ERMS tumors, resulting in decreased expression. However, inhibiting the PI3K/AKT/mTOR pathway has had varied efficacy in RMS. In our RMS mouse model, PTEN localizes to the cytoplasm and nucleus, suggesting that PTEN could have functions other than regulating the PI3K/AKT/mTOR pathway. We demonstrate that Pten loss cooperates in RMS tumorigenesis and results in tumors more reflective of human FN-RMS. We show that Pten loss drives expression of the transcription factor PAX7 and identified PAX7 as a dependency in human FN-RMS. Furthermore, Pax7 deletion completely rescues the deleterious effects of Pten loss but also alters tumor cell fate, giving rise to a smooth muscle tumor. Thus, PTEN loss drives the expression of PAX7, a key member of the RMS core regulatory circuitry dictating tumor cell fate. This highlights a synthetic essential relationship between PTEN and PAX7 in FN-RMS tumor maintenance and tumor-fate decisions. Citation Format: Mark E. Hatley, Casey G. Langdon, Katherine E. Gadek, Matthew R. Garcia, Catherine J. Drummond, Jason A. Hanna, Hongjian E. Jin, Jerold E. Rehg. Rhabdomyosarcoma: visions through the looking glass [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA013.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative changes in rats and mice. The purpose of this publication is to provide a standardized nomenclature for classifying changes observed in the hematolymphoid organs, including the bone marrow, thymus, spleen, lymph nodes, mucosa-associated lymphoid tissues, and other lymphoid tissues (serosa-associated lymphoid clusters and tertiary lymphoid structures) with color photomicrographs illustrating examples of the lesions. Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. The nomenclature for these organs is divided into 3 terminologies: descriptive, conventional, and enhanced. Three terms are listed for each diagnosis. The rationale for this approach and guidance for its application to toxicologic pathology are described in detail below.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children with no improvements in treatment options for RMS patients over the past four decades. Therefore, it is critical to understand the fundamental processes underlying RMS tumorigenesis. RMS is divided into two major histologic subtypes - alveolar and embryonal RMS. Nearly all alveolar RMS express oncogenic fusions of PAX3-FOXO1 or PAX7-FOXO1 whereas embryonal RMS are not driven by these fusion proteins. Instead, embryonal or fusion-negative (FN-RMS) are molecularly heterogeneous. Approximately one-third of fusion-negative RMS (FN-RMS) patients have copy number loss of PTEN (phosphatase and tensin homolog), and approximately 90% of tumors are hypermethylated at the PTEN promoter leading to decreased PTEN expression. This indicates a near universal role for PTEN loss in FN-RMS, but the functional role of PTEN is still unclear. To determine PTEN’s function in FN-RMS, we bred Ptenflox alleles into our aP2-Cre;SmoM2 (ASPWT) FN-RMS mouse model to obtain aP2-Cre;SmoM2;Ptenflox/flox mice (ASPcKO). Conditional Pten deletion accelerated tumorigenesis and produced a tumor with a less differentiated histological appearance, much like human FN-RMS. Interestingly, in PtenWT tumors, we found predominant PTEN immunoreactivity within the nucleus suggesting a role for nuclear PTEN in FN-RMS. Transcriptome analyses revealed robust gene expression changes between the ASPWT and ASPcKO tumors. The top overexpressed gene in ASPcKO tumors was Dbx1 (Developing brain homeobox 1), a homeobox transcription factor with no known cancer function but involved in innate behavioral processes such as breathing. We found FN-RMS patient-derived xenografts are dependent on DBX1 expression, and that DBX1 expression is controlled by PAX7 (Paired Box 7). PAX7 is a transcription factor expressed in satellite cells and maintains a de-differentiated state in FN-RMS. PAX7 expression is also increased in our ASPcKO tumors, and we show that human FN-RMS cells are dependent on PAX7 expression for proliferation. This suggests PTEN loss in FN-RMS engages a new transcriptional program necessary for FN-RMS survival. To determine if Pax7 loss can rescue the deleterious effects of Pten loss in our murine FN-RMS model, we deleted both Pten and Pax7 in our aP2-Cre;SmoM2 mice (ASPcKOP7cKO). ASPcKOP7cKO tumor onset kinetics resembled tumors with wild-type PTEN and were negative for skeletal muscle markers MYOD1 and MYOGENIN. However, these tumors were positive for leiomyosarcoma markers smooth muscle actin and CALDESMON. Together, our data suggests PTEN and PAX7 have a synthetic essential relationship in FN-RMS and that PAX7 is a proliferative driver and lineage dependency for FN-RMS tumors. This work also illustrates the power of murine models to unravel the genetic dependencies underlying both tumor maintenance and identity. Citation Format: Casey G. Langdon, Katherine E. Gadek, Matthew R. Garcia, Myron K. Evans, Kristin B. Reed, Madeline Bush, Jason A. Hanna, Catherine J. Drummond, Matthew C. Maguire, Patrick J. Leavey, David Finkelstein, Hongjian Jin, Patrick A. Schreiner, Jerold E. Rehg, Mark E. Hatley. Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma identity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1667.

Immunocompromised mouse strains expressing human transgenes are being increasingly used in biomedical research. The genetic modifications in these mice cause various cellular responses, resulting in histologic features unique to each strain. The NSG-SGM3 mouse strain is similar to the commonly used NSG (NOD scid gamma) strain but expresses human transgenes encoding stem cell factor (also known as KIT ligand), granulocyte-macrophage colony-stimulating factor, and interleukin 3. This report describes 3 histopathologic features seen in these mice when they are unmanipulated or after transplantation with human CD34+ hematopoietic stem cells (HSCs), virally transduced hCD34+ HSCs, or a leukemia patient-derived xenograft. The first feature is mast cell hyperplasia: unmanipulated, naïve mice develop periductular pancreatic aggregates of murine mast cells, whereas mice given the aforementioned human cells develop a proliferative infiltrative interstitial pancreatic mast cell hyperplasia but with human mast cells. The second feature is the predisposition of NSG-SGM3 mice given these human cells to develop eosinophil hyperplasia. The third feature, secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS)–like disease, is the most pronounced in both its clinical and histopathologic presentations. As part of this disease, a small number of mice also have histiocytic infiltration of the brain and spinal cord with subsequent neurologic or vestibular signs. The presence of any of these features can confound accurate histopathologic interpretation; therefore, it is important to recognize them as strain characteristics and to differentiate them from what may be experimentally induced in the model being studied.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li-Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up. SIGNIFICANCE: A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

Rhabdomyosarcoma (RMS) is an embryonal tumor resembling developing skeletal muscle and the most common pediatric soft-tissue sarcoma. RMS is molecularly defined by the presence or absence of a fusion oncoprotein corresponding with the histological subtypes alveolar and embryonal RMS, respectively. Embryonal, or fusion-negative, RMS (FN-RMS) is heterogeneous in its molecular alteration profile; the major exception is the near universal PTEN promoter hypermethylation found in FN-RMS corresponding with decreased PTEN expression. PTEN's functional role in FN-RMS remains unclear as does PTEN's role in defining tumor fate decisions. Organismal cancer models can help elucidate these decisions by defining the potential tumor fate landscape that can occur in transformed multipotent progenitor cells. Our laboratory leverages a highly penetrant, early onset model of FN-RMS driven by the transdifferentiation of endothelial progenitors into skeletal muscle-like RMS cells by Hedgehog pathway activation. Therefore, our model is uniquely primed to empirically determine the core regulatory factors critical in FN-RMS initiation. Here, we conditionally deleted Pten in these cells (ASPcKO). ASPcKO tumors presented earlier than wild-type tumors and more closely resemble human FN-RMS with a less differentiated skeletal muscle state. These were unique characteristics of ASPcKO tumors as mice with homozygous loss of other tumor suppressors - Cdkn2a, Trp53, and Rb1 - did not recapitulate these phenotypes. We further probed the downstream transcriptional outputs of ASPcKO tumors revealing a profound increase in expression of the neural developmental transcription factors Dbx1 and Pax7. These outputs are functionally important as human FN-RMS patient-derived xenografts are dependent on both DBX1 and PAX7. Subsequently, we also show that DBX1 is a downstream transcriptional target of PAX7 highlighting how Pten loss engages a unique transcriptional program for tumor maintenance. PAX7 is also a core FN-RMS regulatory circuit component. To further interrogate the role of PAX7 on tumor initiation, we concomitantly deleted Pten and Pax7 in our FN-RMS model and found not only that Pax7 loss rescues the survival kinetics observed when Pten is lost, but also alters the developmental trajectory of the tumors that do develop. Instead of Smoothened trans-differentiating our aP2-Cre expressing primordial endothelial cell into a skeletal-muscle like FN-RMS, Pten and Pax7 loss dictates these endothelial cells to give rise to tumors with smooth muscle-like differentiation, including human-like leiomyosarcoma. Together, this synthetic essential interaction between Pten and Pax7 in FN-RMS stresses the importance of the bifunctional role of PAX7 in tumor initiation and maintenance and how specific tumor suppressor loss can engage developmental transcriptional programs to alter tumor fate. Citation Format: Casey G. Langdon, Katherine E. Gadek, Matthew R. Garcia, Kristin B. Reed, Madeline Bush, Jason A. Hanna, Catherine J. Drummond, Matthew C. Maguire, Patrick J. Leavey, David Finkelstein, Hongjian Jin, Jerold E. Rehg, Mark E. Hatley. Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma indentity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3022.

Group3 medulloblastoma (MBG3) that predominantly occur in young children are usually associated with MYC amplification and/or overexpression, frequent metastasis and a dismal prognosis. Physiologically relevant MBG3 models are currently lacking, making inferences related to their cellular origin thus far limited. Using in utero electroporation, we here report that MBG3 mouse models can be developed in situ from different multipotent embryonic cerebellar progenitor cells via conditional expression of Myc and loss of Trp53 function in several Cre driver mouse lines. The Blbp-Cre driver that targets embryonic neural progenitors induced tumors exhibiting a large-cell/anaplastic histopathology adjacent to the fourth ventricle, recapitulating human MBG3. Enforced co-expression of luciferase together with Myc and a dominant-negative form of Trp53 revealed that GABAergic neuronal progenitors as well as cerebellar granule cells give rise to MBG3 with their distinct growth kinetics. Cross-species gene expression analysis revealed that these novel MBG3 models shared molecular characteristics with human MBG3, irrespective of their cellular origin. We here developed MBG3 mouse models in their physiological environment and we show that oncogenic insults drive this MB subgroup in different cerebellar lineages rather than in a specific cell of origin.

Neuroblastoma (NBL) is an embryonal cancer of the sympathetic nervous system (SNS), which causes 15% of pediatric cancer deaths. High-risk NBL is characterized by N-Myc amplification and segmental chromosomal gains and losses. Owing to limited disease models, the etiology of NBL is largely unknown, including both the cell of origin and the majority of oncogenic drivers. We have established a novel system for studying NBL based on the transformation of neural crest cells (NCCs), the progenitor cells of the SNS, isolated from mouse embryonic day 9.5 trunk neural tube explants. Based on pathology and gene expression analysis, we report the first successful transformation of wild-type NCCs into NBL by enforced expression of N-Myc, to generate phenotypically and molecularly accurate tumors that closely model human MYCN-amplified NBL. Using comparative genomic hybridization, we found that NCC-derived NBL tumors acquired copy number gains and losses that are syntenic to those observed in human MYCN-amplified NBL including 17q gain, 2p gain and loss of 1p36. When p53-compromised NCCs were transformed with N-Myc, we generated primitive neuroectodermal tumors with divergent differentiation including osteosarcoma. These subcutaneous tumors were metastatic to regional lymph nodes, liver and lung. Our novel experimental approach accurately models human NBL and establishes a new system with potential to study early stages of NBL oncogenesis, to functionally assess NBL oncogenic drivers and to characterize NBL metastasis.

In humans and in mouse models, precursor B-cell lymphoblastic leukemia (B-ALL)/lymphoblastic lymphoma (B-LBL) can be classified as either the pro-B or pre-B subtype. This is based on the expression of antigens associated with the pro-B and pre-B stages of B-cell development. Antigenic markers can be detected by flow cytometry or immunohistochemistry (IHC), but no comparison of results from these techniques has been reported for murine B-ALL/LBL. In our analysis of 30 cases induced by chemical or viral mutagenesis on a WT or Pax5+/–background, 18 (60%) were diagnosed as pro-B by both flow cytometry and IHC. Discordant results were found for 12 (40%); 6 were designated pro-B by IHC and pre-B by flow cytometry and the reverse for the remaining 6 cases. Discordance occurred because different markers were used to define the pro-B–to–pre-B transition by IHC vs flow cytometry. IHC expression of cytoplasmic IgM (μIgM) defined the pre-B stage, whereas the common practice of using CD25 as a surrogate marker in flow cytometry was employed here. These results show that CD25 and μIgM are not always concurrently expressed in B-ALL/LBL, in contrast to normal B-cell development. Therefore, when subtyping B-ALL/LBL in mice, an IHC panel of B220, PAX5, TdT, c-Kit/CD117, CD43, IgM, and ΚLC should be considered. For flow cytometry, cytoplasmic IgM may be an appropriate marker in conjunction with the surface markers B220, CD19, CD43, c-Kit/CD117, BP-1, and CD25.

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness.

Four distinct subgroups of cerebellar medulloblastomas (MBs) differ in their histopathology, molecular profiles, and prognosis. c-Myc (Myc) or MycN overexpression in granule neuron progenitors (GNPs) induces Group 3 (G3) or Sonic Hedgehog (SHH) MBs, respectively. Differences in Myc and MycN transcriptional profiles depend, in part, on their interaction with Miz1, which binds strongly to Myc but not MycN, to target sites on chromatin. Myc suppresses ciliogenesis and “reprograms” the transcriptome of SHH-dependent GNPs through Miz1-dependent gene repression to maintain “stemness”. Genetic disruption of the Myc/Miz1 interaction inhibited G3 MB development. Target genes of Myc/Miz1 are repressed in human G3 MBs, but not in other subgroups. Therefore, the Myc/Miz1 interaction is a defining hallmark of G3 MB development.