High-energy-demanding tissues, such as skeletal muscle, rely on mitochondrial proteostasis for proper function. Two key quality-control mechanisms -the ubiquitin proteasome system (UPS) and the release of mitochondria-derived vesicles- help maintain mitochondrial proteostasis, but whether these processes interact remains unclear. Here, we show that CRL5Ozz and its substrate, Alix, localize to mitochondria and together regulate the levels and distribution of the mitochondrial solute carrier Slc25A4, which is essential for ATP production. In Ozz−/− or Alix−/− mice, skeletal muscle mitochondria exhibit similar morphological abnormalities, including swelling and dysmorphism, along with partially overlapping metabolomic alterations. We demonstrate that CRL5Ozz ubiquitinates Slc25A4, targeting it for proteasomal degradation, while Alix facilitates Slc25A4 loading into exosomes for lysosomal degradation. Loss of Ozz or Alix in vivo disrupts the steady-state levels of Slc25A4, impairing mitochondrial metabolism and triggering a switch in muscle fiber composition from oxidative, mitochondria-rich slow to glycolytic fast fibers. In skeletal muscle mitochondria, CRL5Ozz and Alix regulate the levels of the ATP transporter Slc25A4; loss of either protein in mice alters Slc25A4, causing mitochondrial defects, metabolic changes, and a shift from oxidative to glycolytic fibers.

The mechanistic target of rapamycin serine/threonine kinase (mTOR), a critical regulator of cell proliferation, is implicated in many human diseases, including cancer. Our laboratory recently discovered a new mTOR complex, called mTORC3, distinct from the two canonical mTOR complexes, mTORC1, and mTORC2, which causes accelerated proliferation and increased malignancy of cancer cells. Understanding how mTORC3 is controlled is an important approach to designing cancer therapies targeting mTORC3. Having investigated how mTORC3 activity was affected by glycogen synthase kinase-3β (GSK3β), a known regulator of mTOR activity, we found that cells do not assemble mTORC3 upon GSK3β knockout, due to extinguished expression of ETV7, an essential component of the mTORC3 complex. We discovered that the lack of ETV7 expression resulted from reduced phosphorylation of serine 727 (S727) of the Signal Transducer and Activator of Transcription 1 (STAT1), which is required for transcriptional activation of ETV7. Finally, we identified that GSK3β through PKCδ phosphorylates STAT1-S727. Together, we identified a novel GSK3β/PKCδ/ STAT1 pathway in Karpas cells that regulates ETV7 transcription, thus positively regulating mTORC3 activity. The identification of this signaling pathway could benefit the therapeutic design for numerous diseases including cancer.### Competing Interest StatementThe authors have declared no competing interest.

mTOR plays a crucial role in cell growth by controlling ribosome biogenesis, metabolism, autophagy, mRNA translation, and cytoskeleton organization. It is a serine/threonine kinase that is part of two distinct extensively described protein complexes, mTORC1 and mTORC2. We have identified a rapamycin resistant mTOR complex, called mTORC3, which is different from the canonical mTORC1 and mTORC2 complexes in that it does not contain the Raptor, Rictor, or mLST8 mTORC1 / 2 components. mTORC3 phosphorylates mTORC1 and mTORC2 targets and contains the ETS transcription factor ETV7, which binds to mTOR and is essential for mTORC3 assembly in the cytoplasm. Tumor cells that assemble mTORC3 have a proliferative advantage and become resistant to rapamycin, indicating that inhibiting mTORC3 may have a therapeutic impact on cancer. Here, we investigate which domains or amino acid residues of ETV7 and mTOR are involved in their mutual binding. We found that the mTOR FRB and LBE sequences in the kinase domain interact with the pointed (PNT) and ETS domains of ETV7, respectively. We also found that forced expression of the mTOR FRB domain in the mTORC3 expressing, rapamycin-resistant cell line Karpas-299 out competes mTOR for ETV7 binding and renders these cells rapamycin-sensitive in vivo. Our data provide useful information for the development of molecules that prevent the assembly of mTORC3, which may have therapeutic value in the treatment of mTORC3 positive cancer.

Supplementary Table 1 from Overexpression of N-Myc Rapidly Causes Acute Myeloid Leukemia in Mice

Supplementary Table 3 from Overexpression of N-Myc Rapidly Causes Acute Myeloid Leukemia in Mice

Supplementary Figure 1 from Overexpression of the Cellular DEK Protein Promotes Epithelial Transformation In vitro and In vivo

SUMMARY High energy-demanding tissues, such as skeletal muscle, require mitochondrial proteostasis to function properly. Two quality-control mechanisms, the ubiquitin proteasome system (UPS) and the release of mitochondria-derived vesicles, safeguard mitochondrial proteostasis. However, whether these processes interact is unknown. Here we show that the E3 ligase CRL5 Ozz , a member of the UPS, and its substrate Alix control the mitochondrial concentration of Slc25A4, a solute carrier that is essential for ATP production. The mitochondria in Ozz −/− or Alix −/− skeletal muscle share overt morphologic alterations (they are supernumerary, swollen, and dysmorphic) and have abnormal metabolomic profiles. We found that CRL5 Ozz ubiquitinates Slc25A4 and promotes its proteasomal degradation, while Alix facilitates SLC25A4 loading into exosomes destined for lysosomal destruction. The loss of Ozz or Alix offsets steady-state levels of Slc25A4, which disturbs mitochondrial metabolism and alters muscle fiber composition. These findings reveal hitherto unknown regulatory functions of Ozz and Alix in mitochondrial proteostasis.

Supplementary Figure 2 from Overexpression of N-Myc Rapidly Causes Acute Myeloid Leukemia in Mice

Supplementary Table 4 from Overexpression of N-Myc Rapidly Causes Acute Myeloid Leukemia in Mice

PDf file - 240K, Expression levels of ectopically expressed proteins (A) Western blot showing exogenous expression of PAX3 and PAX3-FOXO1 (PF) from MSCV-IRES-GFP and MSCV-SV40-Puro retroviral vectors as detected by anti-PAX3 antibody. An anti-Pan actin blot is provided for a loading control. (B) (i)Exogenous expression of PAX3 and PAX3-FOXO1 (PF) in wild type (WT) and Cb1-/- primary myoblasts as detected with the anti-PAX3 antibody. Pan-actin is provided as a loading control. (ii) Real time PCR for Cnr1 mRNA expression in wild type (WT) and Cb1-/- primary myoblasts, normalized to GAPDH. (C) Real time PCR for Cnr1 mRNA expression comparing the relative expression of the Cnr1 and Cnr1 417 mutant constructs.

Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3-Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1(+) RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1-governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1(+) RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1-related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.

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 +/− /ETV7 TG/+/− 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.

During primary tumorigenesis isolated cancer cells may undergo genetic or epigenetic changes that render them responsive to additional intrinsic or extrinsic cues, so that they enter a transitional state and eventually acquire an aggressive, metastatic phenotype. Among these changes is the alteration of the cell metabolic/catabolic machinery that creates the most permissive conditions for invasion, dissemination, and survival. The lysosomal system has emerged as a crucial player in this malignant transformation, making this system a potential therapeutic target in cancer. By virtue of their ubiquitous distribution in mammalian cells, their multifaced activities that control catabolic and anabolic processes, and their interplay with other organelles and the plasma membrane (PM), lysosomes function as platforms for inter- and intracellular communication. This is due to their capacity to adapt and sense nutrient availability, to spatially segregate specific functions depending on their position, to fuse with other compartments and with the PM, and to engage in membrane contact sites (MCS) with other organelles. Here we review the latest advances in our understanding of the role of the lysosomal system in cancer progression. We focus on how changes in lysosomal nutrient sensing, as well as lysosomal positioning, exocytosis, and fusion perturb the communication between tumor cells themselves and between tumor cells and their microenvironment. Finally, we describe the potential impact of MCS between lysosomes and other organelles in propelling cancer growth and spread.

Coordinated regulation of the lysosomal and autophagic systems ensures basal catabolism and normal cell physiology, and failure of either system causes disease. Here we describe an epigenetic rheostat orchestrated by c-MYC and histone deacetylases that inhibits lysosomal and autophagic biogenesis by concomitantly repressing the expression of the transcription factors MiT/TFE and FOXH1, and that of lysosomal and autophagy genes. Inhibition of histone deacetylases abates c-MYC binding to the promoters of lysosomal and autophagy genes, granting promoter occupancy to the MiT/TFE members, TFEB and TFE3, and/or the autophagy regulator FOXH1. In pluripotent stem cells and cancer, suppression of lysosomal and autophagic function is directly downstream of c-MYC overexpression and may represent a hallmark of malignant transformation. We propose that, by determining the fate of these catabolic systems, this hierarchical switch regulates the adaptive response of cells to pathological and physiological cues that could be exploited therapeutically.

The ETS transcription factor ETV7 has been characterized as a hematopoietic oncoprotein, which requires cooperating mutations for its leukemogenic activity. Although the ETV7 gene is highly conserved among vertebrates, part of the rodents, including Mus musculus, deleted the Etv7 gene locus. Many human hematopoietic malignancies upregulate ETV7 expression but contrary to ETV7’s role in oncogenesis, its physiological role in normal tissues is unknown. To determine the physiological function of ETV7 in vivo and determine its role in tumorigenesis in a mouse model, we have generated an ETV7 transgenic mouse that carries a single copy of human BAC DNA containing the ETV7 gene locus and its regulatory sequences. ETV7 heterozygous (ETV7Tg+/WT) mice were fertile, normal in size and born at a normal Mendelian frequency. They had a normal blood count, did not display any gross physical or behavioral abnormalities, and were not tumor-prone. The ETV7 expression pattern in hematopoietic cells of ETV7Tg+/WT mice is very similar to that in human hematopoietic cells. To examine the oncogenic potential of ETV7 in vivo, we crossed ETV7Tg+/WT mice with tumor-prone mouse models. ETV7 greatly accelerated loss of Pten (phosphatase and tensin homolog)-evoked leukemogenesis in PtenΔ/ΔETV7Tg+/WT mice after deletion of the conditional Pten allele. Consistent with this observation, ETV7 expression enhanced the colony-forming and self-renewal activities of primary myeloid Pten−/− cells. In this study we established a transgenic mouse in which we can more accurately model ETV7-associated human tumorigenesis in vivo.

Downstream of insulin-like growth factor receptor, the TSC1/2/ TCB1D7 (tuberous sclerosis complex) and mTOR (mechanistic target of rapamycin) pathways are implicated in many human diseases, including cancer and diabetes. Targeting this pathway is currently an important approach for palliating or eradicating cancer. Downstream of mTOR, translational machinery targeting holds great promise for anticancer drug development. Therefore, we investigated whether the protein synthesis machinery that is regulated by mTORC1 (mTOR complex 1) signaling can in turn regulate mTORC1 activity. We found that inhibition of protein synthesis results in rapid activation of mTORC1 signaling, thereby uncovering a feedback loop between mTOR and the translation machinery. This mTORC1 activation requires tuberous sclerosis complex (TSC) but is independent of AKT. In addition, by using a PKC-δ (protein kinase c delta)-specific inhibitor and PKC-δ siRNA knockdown, we found that PKC-δ kinase activity is required for mTORC1 activation in response to translation inhibitors. Furthermore, translation inhibition activates PKC-δ. Subsequently, we investigated whether PKC-δ can phosphorylate and inactivate TSC1/2, leading to mTORC1 activation. In vitro kinase assays showed direct phosphorylation of TSC2 (S932 and S939) by PKC-δ, which was confirmed by mass spectrometry. In vivo kinase analysis further indicated that both S932 and S939 are phosphorylated in response to translation inhibitors. Finally, phosphorylation defective TSC2 mutants (S932A and S939A single mutants and a S932A/S939A double mutant) failed to upregulate mTORC1 activity in the presence of translation inhibitors, suggesting that activation of mTORC1 by translation inhibitors is mediated by PKC-δ phosphorylation of TSC2 at S932/S939, which inactivates TSC.

The mechanistic target of rapamycin (mTOR) serine/threonine kinase, a critical regulator of cell proliferation, is frequently deregulated in human cancer. Although rapamycin inhibits the two canonical mTOR complexes, mTORC1 and mTORC2, it often shows minimal benefit as an anticancer drug. This is caused by rapamycin resistance of many different tumors, and we show that a third mTOR complex, mTORC3, contributes to this resistance. The ETS (E26 transformation-specific) transcription factor ETV7 interacts with mTOR in the cytoplasm and assembles mTORC3, which is independent of ETV7's transcriptional activity. This complex exhibits bimodal mTORC1/2 activity but is devoid of crucial mTORC1/2 components. Many human cancers activate mTORC3 at considerable frequency, and tumor cell lines that lose mTORC3 expression become rapamycin-sensitive. We show mTORC3's tumorigenicity in a rhabdomyosarcoma mouse model in which transgenic ETV7 expression accelerates tumor onset and promotes tumor penetrance. Discovery of mTORC3 represents an mTOR paradigm shift and identifies a novel target for anticancer drug development.

The increased heme biosynthesis long observed in leukemia was previously of unknown significance. Heme, synthesized from porphyrin precursors, plays a central role in oxygen metabolism and mitochondrial function, yet little is known about its role in leukemogenesis. Here, we show increased expression of heme biosynthetic genes, including UROD, only in pediatric AML samples that have high MYCN expression. High expression of both UROD and MYCN predicts poor overall survival and unfavorable outcomes in adult AML. Murine leukemic progenitors derived from hematopoietic progenitor cells (HPCs) overexpressing a MYCN cDNA (MYCN-HPCs) require heme/porphyrin biosynthesis, accompanied by increased oxygen consumption, to fully engage in self-renewal and oncogenic transformation. Blocking heme biosynthesis reduced mitochondrial oxygen consumption and markedly suppressed self-renewal. Leukemic progenitors rely on balanced production of heme and heme intermediates, the porphyrins. Porphyrin homeostasis is required because absence of the porphyrin exporter, ABCG2, increased death of leukemic progenitors in vitro and prolonged the survival of mice transplanted with Abcg2-KO MYCN-HPCs. Pediatric AML patients with elevated MYCN mRNA display strong activation of TP53 target genes. Abcg2-KO MYCN-HPCs were rescued from porphyrin toxicity by p53 loss. This vulnerability was exploited to show that treatment with a porphyrin precursor, coupled with the absence of ABCG2, blocked MYCN-driven leukemogenesis in vivo, thereby demonstrating that porphyrin homeostasis is a pathway crucial to MYCN leukemogenesis.

Overexpression of DEK oncogene is associated with increased proliferation of carcinoma cells and it is observed in several solid tumors due to the amplification of the 6p22.3 chromosomal region where DEK locates. Although the same chromosomal amplification occurs in multiple myeloma (MM), a plasma cell neoplasm, whether the expression and the copy number of the DEK gene are affected in MM remains elusive. We show that despite the increased copy number in CD138positive MM cells (4 out of 41 MM samples), DEK mRNA expression was down-regulated compared with that in CD138negative bone marrow (BM) cells of the same patients (P<0.0001). DEK protein was not detectable by immunohistochemistry (IHC) in CD138positive normal plasma cells or in malignant plasma cells of MM patients (n = 56) whereas it was widely expressed in normal and neoplastic B-cells. Stable knockdown or overexpression of DEK in CD138positive MM cell lines did not affect the proliferation and viability of the cells profoundly in the presence or absence of chemotherapeutic agent melphalan whereas knockdown of DEK moderately but significantly increased the expression level of CD138 (p<0.01). Decreased DEK expression in plasma cells suggests a potential role of this gene in plasma cell development and lack of detectable DEK protein by IHC could be used as a biomarker for normal and malignant plasma cells.

Many genes in the heme biosynthetic pathway are upregulated in N-MYC-expressing myeloid leukemias; however, it is unknown if or how the heme pathway contributes to leukemogenesis. However, we suspect this pathway is important because one gene in this pathway (UROD) predicts poor overall survival in human myeloid leukemia. The ABC transporter, ABCG2, is a bona fide target of N-MYC and exports toxic heme biosynthetic intermediates (porphyrins). We investigated the role of the heme pathway in leukemic progenitor self-renewal and leukemogenesis. Hematopoietic progenitors from wildtype (WT) mice were retro-virally transduced with an N-MYC expression vector, and leukemic self-renewal was monitored, in vitro, using clonogenic assays. The self-renewal of WT N-MYC-driven leukemic progenitors was reduced by a specific inhibitor of heme biosynthesis, succinylacetone. Importantly, by combining heme with succinylacetone, self-renewal was restored. N-MYC-driven self-renewal was also lost in the absence of Abcg2, which is mostly restored by re-introduction of ABCG2. Collectively, these findings indicate N-MYC driven leukemic progenitors require a balance in the level of heme and the intermediates in this biosynthetic pathway. The impaired survival of Abcg2-deficient leukemic progenitors is associated with increased mitochondrial ROS and the loss of viability of leukemic progenitors. We further demonstrated that absence of Abcg2 disrupted N-MYC leukemic progression and that over-accumulation of porphyrins appears to account for the blocked leukemogenesis in vivo. We propose that modulating this pathway provides another therapeutic approach for treating myeloid leukemias that express high levels of N-MYC. This work was supported by NIH and by the ALSAC.