ClpX functions as either an independent chaperone or a component of the ClpXP protease, a conserved intracellular protease that acts as a global regulator in the bacterial cell by degrading regulatory proteins, stress response proteins and rate-limiting enzymes. Previously, we found that loss of clpX in Bacillus anthracis Sterne leads to increased susceptibility to antimicrobial agents that target the cell envelope. The aim of this study was to identify genes within the regulatory network of clpX that contribute to antimicrobial resistance. Using microarray analysis, we found 119 genes that are highly differentially expressed in the ∆clpX mutant, with the majority involved in metabolic, transport or regulatory functions. Several of these differentially expressed genes, including glpF, sigM, mrsA, lrgA and lrgB, are associated with cell wall-active antibiotics in other bacterial species. We focused on lrgA and lrgB, which form the lrgAB operon and are downregulated in ∆clpX, because loss of lrgAB increases autolytic activity and penicillin susceptibility in Staphylococcus aureus. While we observed no changes in autolytic activity in either ∆clpX or ∆lrgAB B. anthracis Sterne, we find that both mutants have increased susceptibility to the antimicrobial peptide LL-37 and daptomycin. However, phenotypes between ∆clpX and ∆lrgAB are not identical as ∆clpX also displays increased susceptibility to penicillin and nisin but ∆lrgAB does not. Therefore, while decreased expression of lrgAB may be partially responsible for the increased antimicrobial susceptibility seen in the ∆clpX mutant, disruption of other pathways must also contribute to this phenotype.
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.
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.
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite aggressive chemotherapy, radiotherapy, and surgery, 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, salivary gland, biliary tree, and omentum. 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 that display the histologic and molecular characteristics of human embryonal RMS (ERMS). With the short latency and anatomically restricted tumor location in the neck, we sought to leverage this model to explore the cell of origin of ERMS. Lineage tracing experiments identified aP2-Cre labeled cells are distinctly nonmyogenic and were identified as endothelial cells found in the interstitium between muscle fibers. We illustrate that aP2-Cre is not expressed in the quiescent or activated muscle stem cells or satellite cells. Expression of oncogenic SmoM2 with aP2-Cre results in proliferation and expansion of the aP2-Cre labeled muscle interstitial endothelial cells and myogenic transdifferentiation resulting in ERMS. Activation of the hedgehog pathway in aP2-Cre labeled endothelial progenitors results in expression of skeletal muscle specification factors specific in the head and neck development, including TBX1, PITX2, TCF21, and MSC. We illustrate that endothelium and skeletal muscle within the head and neck arise from KDR (VEFGR2)-expressing progenitors. Hedgehog pathway activation in committed KDR+ endothelial progenitors results in Tbx1 expression and subsequent MYOD1 expression driving a partially myogenic program characteristic of ERMS. 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. Citation Format: Catherine J. Drummond, Kathrine E. Gadek, Madeline Bush, Matthew R. Garcia, Mark E. Hatley. Developmental reprogramming via Hedgehog pathway activation in nonmyogenic endothelial progenitors drives fusion-negative rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR01.
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.
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