Haploinsufficiency of the lysosomal sialidase NEU1 results in a model of pleomorphic rhabdomyosarcoma in mice.

Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1+/-/ETV7TG/+/- mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1. These tumors share mostly features of embryonal and some of alveolar rhabdomyosarcoma. Mechanistically, we show that the transforming pathway is increased lysosomal exocytosis downstream of reduced neuraminidase 1, exemplified by the redistribution of the lysosomal associated membrane protein 1 at the plasma membrane of tumor and stromal cells. Here we exploit this unique feature for single cell analysis and define heterogeneous populations of exocytic, only partially differentiated cells that force tumors to pleomorphism and promote a fibrotic microenvironment. These data together with the identification of an adipogenic signature shared by human rhabdomyosarcoma, and likely fueling the tumor's metabolism, make this model of pleomorphic rhabdomyosarcoma ideal for diagnostic and therapeutic studies.

Requirement for antiapoptotic MCL-1 during early erythropoiesis.

Although BCL-xL is critical to the survival of mature erythrocytes, it is still unclear whether other antiapoptotic molecules mediate survival during earlier stages of erythropoiesis. Here, we demonstrate that erythroid-specific Mcl1 deletion results in embryonic lethality beyond embryonic day 13.5 as a result of severe anemia caused by a lack of mature red blood cells (RBCs). Mcl1-deleted embryos exhibit stunted growth, ischemic necrosis, and decreased RBCs in the blood. Furthermore, we demonstrate that MCL-1 is only required during early definitive erythropoiesis; during later stages, developing erythrocytes become MCL-1 independent and upregulate the expression of BCL-xL. Functionally, MCL-1 relies upon its ability to prevent apoptosis to promote erythroid development because codeletion of the proapoptotic effectors Bax and Bak can overcome the requirement for MCL-1 expression. Furthermore, ectopic expression of human BCL2 in erythroid progenitors can compensate for Mcl1 deletion, indicating redundancy between these 2 antiapoptotic family members. These data clearly demonstrate a requirement for MCL-1 in promoting survival of early erythroid progenitors.

Resolving medulloblastoma cellular architecture by single-cell genomics.

Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

Efficacy and toxicity of a virus-directed enzyme prodrug therapy purging method: preclinical assessment and application to bone marrow samples from neuroblastoma patients.

Autologous stem cell transplantation is used to rescue cancer patients from myelosuppression caused by high-dose chemotherapy. However, autologous grafts often contain tumor cells that can contribute directly to relapse. Current purging methods are useful when fewer than 1% tumor cells contaminate the bone marrow, and patients with tumor burdens of >1% are considered ineligible for chemotherapy that necessitates stem cell rescue. Using neuroblastoma (NB) as a model system, we developed a method that is effective even with tumor burdens of 10-25%. Mixtures of NB-1691 NB cells and CD34(+) hematopoietic cells purged by this method showed no evidence of viable tumor cells as assessed by clonogenic assays or reverse transcription-PCR for the NB cell markers tyrosine hydroxylase and N-MYC. The efficacy and lack of toxicity of the method were verified using in vivo mouse models. Severe combined immunodeficient mice that received purged cell preparations containing 10% NB-1691 cells survived without evidence of disease for the observation period (>1 year), whereas mice that received unpurged cells developed disseminated disease requiring euthanasia 73-96 days after injection of cells. No evidence of toxicity to the mice was detected by numerous laboratory values for bone marrow, liver, and kidney function, and no difference was seen in the ability of purged cell mixtures versus unmanipulated CD34(+) cells to reconstitute the marrow of non-obese diabetic severe combined immunodeficient mice. In a pilot study, marrow was obtained from eight patients who had >/=1% metastatic tumor burden. All eight samples were purged to the level of detection by reverse transcription-PCR (samples from seven patients) or clonogenic potential (sample from one patient), whichever assay was used. The described adenovirus/rabbit carboxylesterase/CPT-11 (irinotecan, 7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) virus-directed enzyme prodrug method may be useful for patients whose tumor burdens exceed 1% at the time of stem cell harvest. Assessment of purging efficacy with additional samples from NB patients is ongoing.