Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALKF1174L mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALKF1174L/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALKF1174L/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors

Cellular Dynamics of Tympanic Membrane Homeostasis and Perforation Repair

The tympanic membrane (TM) is a crucial component of the conductive apparatus of the ear. It defines the border between the external auditory canal (EAC) and middle ear, and it transmits sound vibrations gathered in the EAC to the first middle ear ossicle, the malleus. There is much important pathology of the TM, including perforations (both acute and chronic), cholesteatoma, keratosis obturans, tympanosclerosis, and myringitis. Our lack of understanding of the basic biology of the TM has hampered our understanding of and ability to treat these conditions. The primary goal of the work presented herein was to elucidate mechanisms underlying TM maintenance and wound response.To catalog the specific cell populations of the TM, we conducted single-cell RNA sequencing of dissociated murine and human TM tissue, followed by validation of the populations by immunofluorescence and in situ hybridization of whole-mount and sectioned tissue. We demonstrated that the keratinocytes of the pars tensa epidermis are largely undifferentiated, and that what differentiation occurs is not obligately paired with stratification as it is at other epidermal sites. Furthermore, we studied turnover of the tissue with continuous EdU uptake, and determined that the entire epidermal layer proliferates in approximately three weeks under homeostatic conditions, but in under one week following a perforation. Additionally, we utilized two Cre drivers, Ki67-CreERT2 and Krt5-CreERT2, paired with the Confetti reporter allele to characterize the clonal architecture of the TM epidermis. The development of clones qualitatively supports the model of neutral drift dynamics in the stem cell pool. Features of individual clones suggests that cells in the proliferative region of the superior pars tensa contribute to long-term maintenance of the tissue and can thus be termed stem cells (SC), while cells in the proliferative region of the malleus only transiently contribute cells to the tissue and can thus be considered more committed progenitors (CP). We propose a model in which these distinct SC and CP populations are maintained by the continuous migration of keratinocytes over the TM. Lastly, we identify Pdgfra signaling in fibroblasts as a molecular signal required to maintain the proliferative niches of the TM

A Hierarchy of Proliferative and Migratory Keratinocytes Maintains the Tympanic Membrane

The tympanic membrane (TM) is critical for hearing and requires continuous clearing of cellular debris, but little is known about homeostatic mechanisms in the TM epidermis. Using single-cell RNA sequencing, lineage tracing, whole-organ explant, and live-cell imaging, we show that homeostatic TM epidermis is distinct from other epidermal sites and has discrete proliferative zones with a three-dimensional hierarchy of multiple keratinocyte populations. TM stem cells reside in a discrete location of the superior TM and generate long-lived clones and committed progenitors (CPs). CP clones exhibit lateral migration, and their proliferative capacity is supported by Pdgfra+ fibroblasts, generating migratory but non-proliferative progeny. Single-cell sequencing of the human TM revealed similar cell types and transcriptional programming. Thus, during homeostasis, TM keratinocytes transit through a proliferative CP state and exhibit directional lateral migration. This work forms a foundation for understanding TM disorders and modeling keratinocyte biology

Targeting MTHFD2 in acute myeloid leukemia

Drugs targeting metabolism have formed the backbone of therapy for some cancers. We sought to identify new such targets in acute myeloid leukemia (AML). The one-carbon folate pathway, specifically methylenetetrahydrofolate dehydrogenase-cyclohydrolase 2 (MTHFD2), emerged as a top candidate in our analyses. MTHFD2 is the most differentially expressed metabolic enzyme in cancer versus normal cells. Knockdown of MTHFD2 in AML cells decreased growth, induced differentiation, and impaired colony formation in primary AML blasts. In human xenograft and MLL-AF9 mouse leukemia models, MTHFD2 suppression decreased leukemia burden and prolonged survival. Based upon primary patient AML data and functional genomic screening, we determined that FLT3-ITD is a biomarker of response to MTHFD2 suppression. Mechanistically, MYC regulates the expression of MTHFD2, and MTHFD2 knockdown suppresses the TCA cycle. This study supports the therapeutic targeting of MTHFD2 in AML. It has been known for decades that cancer cells have an altered metabolism. As early as the 1920s, Otto Warburg observed that tumor cells consume glucose at a high rate and undergo fermentation even in the presence of oxygen (Warburg et al., 1927). Since then, drugs targeting metabolism have transformed the treatment of certain cancers. In the 1940s, the discovery and application of aminopterin, which was later found to target dihydrofolate reductase (DHFR), a cytoplasmic enzyme involved in one-carbon folate metabolism, yielded the first remission in a child with acute lymphoblastic leukemia (Farber et al., 1948). Other folate derivatives, such as methotrexate, were later developed. More recently, drugs such as 5-fluorouracil and pemetrexed that target thymidylate synthetase, another enzyme involved in one-carbon folate metabolism, were found to be effective therapies for some cancers (Locasale, 2013). The discovery of germline and somatic mutations that alter metabolic proteins in cancer further supports the role of altered metabolism in cancer pathogenesis. Mutations in genes of the succinate dehydrogenase complex, critical for both the tricarboxylic acid (TCA) cycle and electron transport chain, have been implicated in the pathogenesis of hereditary paragangliomas (Baysal et al., 2000; Niemann and Müller, 2000), pheochromocytomas (Astuti et al., 2001), renal cell cancer (Vanharanta et al., 2004), and gastrointestinal stromal tumors (Janeway et al., 2011; Pantaleo et al., 2011). In addition, mutations in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) have been found in subsets of gliomas (Yan et al., 2009; Brennan et al., 2013) and acute myeloid leukemia (AML; Paschka et al., 2010; Cancer Genome Atlas Research Network, 2013), among other malignancies. Drugs targeting these mutant proteins have entered the clinic with some successes in early phase trials (Stein et al. 2014. 56th Annual American Hematoligical Society Annual Meeting and Exposition. Abstract 115.). Moreover, as understanding of the metabolic derangements necessary to promote and maintain the malignant state continues to expand, so does the list of potential drug targets. For example, aerobic glycolysis is thought to enable the generation of the nucleotides, proteins, and lipids necessary to maintain the malignant proliferative state, in part through regulation of the glycolytic enzyme pyruvate kinase (Vander Heiden et al., 2010). Additionally, the discovery of the critical importance of glycine and serine in cancer metabolism has led to a resurgence in interest in better understanding the mechanistic relevance of one-carbon folate metabolism (Jain et al., 2012; Zhang et al., 2012; Labuschagne et al., 2014; Ye et al., 2014; Kim et al., 2015; Maddocks et al., 2016). Although drugs targeting metabolism, such as methotrexate and asparaginase (a drug that reduces the availability of asparagine and glutamine), have been critical for the treatment of acute lymphoblastic leukemia, they are not used in therapy for AML, a hematopoietic malignancy where cure rates are still quite poor despite high-dose cytotoxic chemotherapy, including stem cell transplantation. This is especially true for patients with subtypes of AML characterized by high-risk features, such as the presence of FLT3-ITD mutations. New therapies are urgently needed for the treatment of these patients. In this study, we set out to define common mechanisms critical to the maintenance of AML cells to nominate novel, potentially targetable metabolic pathways for the treatment of this disease. We integrated gene expression signatures generated from the treatment of AML cells with multiple small molecules known to promote AML differentiation and death. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), an NAD+-dependent enzyme with dehydrogenase and cyclohydrolase activity, which plays an essential role in mitochondrial one-carbon folate metabolism, was prioritized as a target relevant to AML cell growth and differentiation. Suppression of MTHFD2 impaired AML growth and induced differentiation in vitro and impaired disease progression in multiple mouse models of AML. Additionally, FLT3-ITD mutations are a biomarker of response to MTHFD2 suppression. Mechanistically, MYC directly regulates MTHFD2 expression, and suppression of MTHFD2 leads to marked alteration of the TCA cycle.National Cancer Institute (U.S.) (R01 CA140292)National Cancer Institute (U.S.) (R21 CA198028)National Heart, Lung, and Blood Institute (5T32 HL07574-32)Eunice Kennedy Shriver National Institute of Child Health and Human Development (U.S.) (5K12HD052896-09)Library of Integrated Cellular Signatures (U54HG006093)Library of Integrated Cellular Signatures (U54HL127366

Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition

Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically-defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis demonstrated downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knock-down phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in three in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma

Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition

Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis showed downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knockdown phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma

Targeting MTHFD2 in acute myeloid leukemia

Drugs targeting metabolism have formed the backbone of therapy for some cancers. We sought to identify new such targets in acute myeloid leukemia (AML). The one-carbon folate pathway, specifically methylenetetrahydrofolate dehydrogenase-cyclohydrolase 2 (MTHFD2), emerged as a top candidate in our analyses. MTHFD2 is the most differentially expressed metabolic enzyme in cancer versus normal cells. Knockdown of MTHFD2 in AML cells decreased growth, induced differentiation, and impaired colony formation in primary AML blasts. In human xenograft and MLL-AF9 mouse leukemia models, MTHFD2 suppression decreased leukemia burden and prolonged survival. Based upon primary patient AML data and functional genomic screening, we determined that FLT3-ITD is a biomarker of response to MTHFD2 suppression. Mechanistically, MYC regulates the expression of MTHFD2, and MTHFD2 knockdown suppresses the TCA cycle. This study supports the therapeutic targeting of MTHFD2 in AML

The intersection of genetic and chemical genomic screens identifies GSK-3α as a target in human acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α–specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3β has been well studied in cancer development, these studies support a role for GSK-3α in AML