Jordan M. Blum

Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia

Although the role of Hedgehog (Hh) signalling in embryonic pattern formation is well established, its functions in adult tissue renewal and maintenance remain unclear, and the relationship of these functions to cancer development has not been determined. Here we show that the loss of Smoothened (Smo), an essential component of the Hh pathway, impairs haematopoietic stem cell renewal and decreases induction of chronic myelogenous leukaemia (CML) by the BCR–ABL1 oncoprotein. Loss of Smo causes depletion of CML stem cells—the cells that propagate the leukaemia—whereas constitutively active Smo augments CML stem cell number and accelerates disease. As a possible mechanism for Smo action, we show that the cell fate determinant Numb, which depletes CML stem cells, is increased in the absence of Smo activity. Furthermore, pharmacological inhibition of Hh signalling impairs not only the propagation of CML driven by wild-type BCR–ABL1, but also the growth of imatinib-resistant mouse and human CML. These data indicate that Hh pathway activity is required for maintenance of normal and neoplastic stem cells of the haematopoietic system and raise the possibility that the drug resistance and disease recurrence associated with imatinib treatment of CML might be avoided by targeting this essential stem cell maintenance pathway.

Regulation of myeloid leukaemia by the cell-fate determinant Musashi

Chronic myelogenous leukaemia (CML) can progress from a slow growing chronic phase to an aggressive blast crisis phase, but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML to show that disease progression is regulated by the Musashi–Numb signalling axis. Specifically, we find that the chronic phase is marked by high levels of Numb expression whereas the blast crisis phase has low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced-phase disease in vivo. As a possible explanation for the decreased levels of Numb in the blast crisis phase, we show that NUP98–HOXA9, an oncogene associated with blast crisis CML, can trigger expression of the RNA-binding protein Musashi2 (Msi2), which in turn represses Numb. Notably, loss of Msi2 restores Numb expression and significantly impairs the development and propagation of blast crisis CML in vitro and in vivo. Finally we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi–Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for the therapy of aggressive leukaemias.

Imaging Hematopoietic Precursor Division in Real Time

Stem cells are thought to balance self-renewal and differentiation through asymmetric and symmetric divisions, but whether such divisions occur during hematopoietic development remains unknown. Using a Notch reporter mouse, in which GFP acts as a sensor for differentiation, we image hematopoietic precursors and show that they undergo both symmetric and asymmetric divisions. In addition we show that the balance between these divisions is not hardwired but responsive to extrinsic and intrinsic cues. Precursors in a prodifferentiation environment preferentially divide asymmetrically, whereas those in a prorenewal environment primarily divide symmetrically. Oncoproteins can also influence division pattern: although BCR-ABL predominantly alters the rate of division and death, NUP98-HOXA9 promotes symmetric division, suggesting that distinct oncogenes subvert different aspects of cellular function. These studies establish a system for tracking division of hematopoietic precursors and show that the balance of symmetric and asymmetric division can be influenced by the microenvironment and subverted by oncogenes.

Distinct and Overlapping Sarcoma Subtypes Initiated from Muscle Stem and Progenitor Cells

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, whereas undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue sarcomas diagnosed in adults. To investigate the myogenic cell(s) of origin of these sarcomas, we used Pax7-CreER and MyoD-CreER mice to transform Pax7(+) and MyoD(+) myogenic progenitors by expressing oncogenic Kras(G12D) and deleting Trp53 in vivo. Pax7-CreER mice developed RMS and UPS, whereas MyoD-CreER mice developed UPS. Using gene set enrichment analysis, RMS and UPS each clustered specifically within their human counterparts. These results suggest that RMS and UPS have distinct and overlapping cells of origin within the muscle lineage. Taking them together, we have established mouse models of soft tissue sarcoma from muscle stem and progenitor cells.

Embryonic Signaling Pathways and Rhabdomyosarcoma: Contributions to Cancer Development and Opportunities for Therapeutic Targeting

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence, accounting for approximately 7% of childhood cancers. Current therapies include nonspecific cytotoxic chemotherapy regimens, radiation therapy, and surgery; however, these multimodality strategies are unsuccessful in the majority of patients with high-risk disease. It is generally believed that these tumors represent arrested or aberrant skeletal muscle development, and, accordingly, developmental signaling pathways critical to myogenesis such as Notch, WNT, and Hedgehog may represent new therapeutic targets. In this paper, we summarize the current preclinical studies linking these embryonic pathways to rhabdomyosarcoma tumorigenesis and provide support for the investigation of targeted therapies in this embryonic cancer.

Role of p53 in regulating tissue response to radiation by mechanisms independent of apoptosis

Radiation exposure leads to diverse outcomes in vivo across different tissues and even within the same cell lineage. The diversity of radiation response in vivo is at least partially attributable to the status of the tumor suppressor p53, a master regulator of cellular response to stress, and activation of its transcriptional targets. In certain cells, such as hematopoietic progenitors and transit amplifying cells in the gastrointestinal epithelium, activation of p53 by radiation triggers the intrinsic pathway of apoptosis. However, in many other cells, activation of p53 by radiation does not result in apoptosis, which underscores the importance of understanding the role of p53 in regulating radiation response through alternative mechanisms. In this review, we summarize recent studies using genetically engineered mice to dissect the role of p53 in 1) cells where its activation is dissociated from the intrinsic pathway of apoptosis, such as hematopoietic stem cells and vascular endothelial cells and 2) tissues where activation of the intrinsic pathway of apoptosis does not promote the acute radiation syndrome, such as the gastrointestinal epithelium. We highlight findings showing that the apoptosis-independent response of p53 to radiation in vivo can contribute to death or survival in a cell-type dependent manner, which underscores the complexity by which p53 regulates the cellular and tissue response to radiation.

Acute Tissue Injury Activates Satellite Cells and Promotes Sarcoma Formation via the HGF/c-MET Signaling Pathway

Some patients with soft-tissue sarcoma (STS) report a history of injury at the site of their tumor. Although this phenomenon is widely reported, there are relatively few experimental systems that have directly assessed the role of injury in sarcoma formation. We recently described a mouse model of STS whereby p53 is deleted and oncogenic Kras is activated in muscle satellite cells via a Pax7(CreER) driver following intraperitoneal injection with tamoxifen. Here, we report that after systemic injection of tamoxifen, the vast majority of Pax7-expressing cells remain quiescent despite mutation of p53 and Kras. The fate of these muscle progenitors is dramatically altered by tissue injury, which leads to faster kinetics of sarcoma formation. In adult muscle, quiescent satellite cells will transition into an active state in response to hepatocyte growth factor (HGF). We show that modulating satellite cell quiescence via intramuscular injection of HGF increases the penetrance of sarcoma formation at the site of injection, which is dependent on its cognate receptor c-MET. Unexpectedly, the tumor-promoting effect of tissue injury also requires c-Met. These results reveal a mechanism by which HGF/c-MET signaling promotes tumor formation after tissue injury in a mouse model of primary STS, and they may explain why some patients develop a STS at the site of injury.

Acute DNA damage activates the tumour suppressor p53 to promote radiation-induced lymphoma

Genotoxic cancer therapies, such as chemoradiation, cause haematological toxicity primarily by activating the tumour suppressor p53. While inhibiting p53-mediated cell death during cancer therapy ameliorates haematologic toxicity, whether it also impacts carcinogenesis remains unclear. Here we utilize a mouse model of inducible p53 short hairpin RNA (shRNA) to show that temporarily blocking p53 during total-body irradiation (TBI) not only ameliorates acute toxicity, but also improves long-term survival by preventing lymphoma development. Using KrasLA1 mice, we show that TBI promotes the expansion of a rare population of thymocytes that express oncogenic KrasG12D. However, blocking p53 during TBI significantly suppresses the expansion of KrasG12D-expressing thymocytes. Mechanistically, bone marrow transplant experiments demonstrate that TBI activates p53 to decrease the ability of bone marrow cells to suppress lymphoma development through a non-cell-autonomous mechanism. Together, our results demonstrate that the p53 response to acute DNA damage promotes the development of radiation-induced lymphoma.

Methods to Generate Genetically Engineered Mouse Models of Soft Tissue Sarcoma

We discuss the generation of primary soft tissue sarcomas in mice using the Cre-loxP system to activate conditional mutations in oncogenic Kras and the tumor suppressor p53 (LSL-Kras(G12D/+); p53(flox/flox)). Sarcomas can be generated either by adenoviral delivery of Cre recombinase, activation of transgenic Cre recombinase with tamoxifen, or through transplantation of isolated satellite cells with Cre activation in vitro. Various applications of these models are discussed, including anticancer therapies, metastasis, in vivo imaging, and genetic requirements for tumorigenesis.

Abstract A64: A role for injury in sarcomagenesis

We developed a primary mouse model of embryonal rhabdomyosarcoma and discovered that tissue injury dramatically accelerates sarcoma formation at the site of injury. We were intrigued by this finding as many sarcoma patients anecdotally report a history of trauma prior to sarcoma development. We therefore utilized this mouse model system to explore the mechanism of injury-mediated sarcoma formation. Our preliminary findings suggest that the majority of muscle progenitor cells remain quiescent following mutation of genes with high transformation potential. However, tissue injury “flips a switch”, thereby pushing resting muscle progenitor cells into a proliferative phase. In the setting of a normal mouse, proliferation of muscle cells results in resolution of the injury in a matter of weeks. Conversely, when cells in the vicinity of injury harbor transformative gene mutations, the “switch” does not return to an off-state, and a tumor rapidly develops at the injury site. Our novel mouse model of embryonal rhabdomyosarcoma provides temporospatial control over the deletion of p53 and activation of Kras in muscle progenitor cells. Specifically, Pax7-CreERT2 (P7) mice express a tamoxifen-inducible Cre downstream of the endogenous promoter for the satellite cell transcription factor Pax7. P7 mice were crossed to genetically engineered mice containing a lox-STOP-lox cassette upstream of oncogenic K-ras G12D (K) in addition to 2 floxed p53 alleles (P) to generate P7KP mice. P7KP mice were injected with systemic, intraperitoneal (IP) tamoxifen to mediate Cre-dependent recombination in Pax7-expressing cells. When treated in this manner, sarcomas arise throughout the animal with 100% penetrance with a median onset of 45 days. In contrast, tumor onset is dramatically accelerated when P7KP mice are injured with cardiotoxin, a component of cobra venom that causes myonecrosis. P7KP mice treated with IP tamoxifen along with concurrent intramuscular (IM) cardiotoxin develop sarcomas at the site of injury with a median onset of 15 days. Cardiotoxin was next injected into the gastrocnemius muscle at time points either before, concurrent, or after IP tamoxifen to better define the effect of injury on sarcoma formation. Cardiotoxin promoted highly efficient transformation when administered up to 3 days before and up to 21 days after IP tamoxifen administration. Lineage tracing studies showed no appreciable proliferation of recombined Pax7+ cells in the absence of cardiotoxin, suggesting that sarcoma formation requires factors in addition to loss of p53 and activation of K-ras to cause sarcoma. In order to test the hypothesis that proliferating satellite cells are more prone to sarcoma formation, we treated P7KP mice in the gastrocnemius muscle with hepatocyte growth factor (HGF) along with systemic, IP tamoxifen. HGF has been shown to promote proliferation of resting satellite cells. Interestingly, 12/13 mice treated with IM HGF developed sarcomas at the injection site in a median of 37 days versus 3/12 of mice treated with vehicle control. Current experiments are aimed at dissecting the role of the immune system in the cardiotoxin response. Our experiments support the role of tissue injury acting as a classic promoter in the initiator/promoter model of tumorigenesis. Thus perturbation of the microenvironment has a dramatic effect on sarcoma formation. While the majority of cancer therapeutics are directed against mutations within the tumor, our findings reveal alternative candidates to prevent sarcoma initiation and perhaps maintenance. We anticipate that an understanding of signaling events at the earliest stages of sarcoma formation will provide new drug targets to treat existing tumors and potentially prevent the outgrowth of micrometastases. Citation Format: David Van Mater, Leonor Ano, Jordan Blum, David G. Kirsch. A role for injury in sarcomagenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A64.