Congenital X-linked neutropenia with myelodysplasia and somatic tetraploidy due to a germline mutation in SEPT6.

Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.

Identification and characterization of an alternative cancer-derived PD-L1 splice variant.

Therapeutic blockade of the PD-1/PD-L1 axis is recognized as an effective treatment for numerous cancer types. However, only a subset of patients respond to this treatment, warranting a greater understanding of the biological mechanisms driving immune evasion via PD-1/PD-L1 signaling and other T-cell suppressive pathways. We previously identified a head and neck squamous cell carcinoma with human papillomavirus integration in the PD-L1 locus upstream of the transmembrane domain-encoding region, suggesting expression of a truncated form of PD-L1 (Parfenov et al., Proc Natl Acad Sci USA 111(43):15544-15549, 2014). In this study, we extended this observation by performing a computational analysis of 33 other cancer types as well as human cancer cell lines, and identified additional PD-L1 isoforms with an exon 4 enrichment expressed in 20 cancers and human cancer cell lines. We demonstrate that cancer cell lines with high expression levels of exon 4-enriched PD-L1 generate a secreted form of PD-L1. Further biochemical studies of exon 4-enriched PD-L1 demonstrated that this form is secreted and maintains the capacity to bind PD-1 as well as to serve as a negative regulator on T cell function, as measured by inhibition of IL-2 and IFNg secretion. Overall, we have demonstrated that truncated forms of PD-L1 exist in numerous cancer types, and have validated that truncated PD-L1 can be secreted and negatively regulate T cell function.

The Prochlorococcus carbon dioxide-concentrating mechanism: evidence of carboxysome-associated heterogeneity.

The ability of Prochlorococcus to numerically dominate open ocean regions and contribute significantly to global carbon cycles is dependent in large part on its effectiveness in transforming light energy into compounds used in cell growth, maintenance, and division. Integral to these processes is the carbon dioxide-concentrating mechanism (CCM), which enhances photosynthetic CO2 fixation. The CCM involves both active uptake systems that permit intracellular accumulation of inorganic carbon as the pool of bicarbonate and the system of HCO3 (-) conversion into CO2. The latter is located in the carboxysome, a microcompartment designed to promote the carboxylase activity of Rubisco. This study presents a comparative analysis of several facets of the Prochlorococcus CCM. Our analyses indicate that a core set of CCM components is shared, and their genomic organization is relatively well conserved. Moreover, certain elements, including carboxysome shell polypeptides CsoS1 and CsoS4A, exhibit striking conservation. Unexpectedly, our analyses reveal that the carbonic anhydrase (CsoSCA) and CsoS2 shell polypeptide have diversified within the lineage. Differences in csoSCA and csoS2 are consistent with a model of unequal rates of evolution rather than relaxed selection. The csoS2 and csoSCA genes form a cluster in Prochlorococcus genomes, and we identified two conserved motifs directly upstream of this cluster that differ from the motif in marine Synechococcus and could be involved in regulation of gene expression. Although several elements of the CCM remain well conserved in the Prochlorococcus lineage, the evolution of differences in specific carboxysome features could in part reflect optimization of carboxysome-associated processes in dissimilar cellular environments.

ZBTB33 is mutated in clonal hematopoiesis and myelodysplastic syndromes and impacts RNA splicing.

Clonal hematopoiesis results from somatic mutations in cancer driver genes in hematopoietic stem cells. We sought to identify novel drivers of clonal expansion using an unbiased analysis of sequencing data from 84,683 persons and identified common mutations in the 5-methylcytosine reader, ZBTB33, as well as in YLPM1, SRCAP, and ZNF318. We also identified these mutations at low frequency in myelodysplastic syndrome patients. Zbtb33 edited mouse hematopoietic stem and progenitor cells exhibited a competitive advantage in vivo and increased genome-wide intron retention. ZBTB33 mutations potentially link DNA methylation and RNA splicing, the two most commonly mutated pathways in clonal hematopoiesis and MDS.

Characterization of DDR2 Inhibitors for the Treatment of DDR2 Mutated Nonsmall Cell Lung Cancer.

Despite advances in precision medicine approaches over the past decade, the majority of nonsmall cell lung cancers (NSCLCs) are refractory to treatment with targeted small molecule inhibitors. Previous work has identified mutations in the Discoidin Domain Receptor 2 (DDR2) kinase as potential therapeutic targets in NSCLCs. While DDR2 is potently targeted by several multitargeted kinase inhibitors, most notably dasatinib, toxicity has limited the clinical application of anti-DDR2 therapy. Here, we have characterized compound 1 and other tool compounds demonstrating selectivity for DDR2 and show that while these compounds inhibit DDR2 in lung cancer model systems, they display limited antiproliferative activity in DDR2 mutated cell lines as compared to dual DDR2/SRC inhibitors. We show that DDR2 and SRC are binding partners, that SRC activity is tied to DDR2 activation, and that dual inhibition of both DDR2 and SRC leads to enhanced suppression of DDR2 mutated lung cancer cell lines. These results support the further evaluation of dual SRC/DDR2 targeting in NSCLC, and we report a tool compound, compound 5, which potently inhibits both SRC and DDR2 with a distinct selectivity profile as compared to dasatinib.

NSCLC Driven by DDR2 Mutation Is Sensitive to Dasatinib and JQ1 Combination Therapy.

Genetically engineered mouse models of lung cancer have demonstrated an important role in understanding the function of novel lung cancer oncogenes and tumor-suppressor genes identified in genomic studies of human lung cancer. Furthermore, these models are important platforms for preclinical therapeutic studies. Here, we generated a mouse model of lung adenocarcinoma driven by mutation of the discoidin domain receptor 2 (DDR2) gene combined with loss of TP53. DDR2(L63V);TP53(L/L) mice developed poorly differentiated lung adenocarcinomas in all transgenic animals analyzed with a latency of 40 to 50 weeks and a median survival of 67.5 weeks. Mice expressing wild-type DDR2 with combined TP53 loss did not form lung cancers. DDR2(L63V);TP53(L/L) tumors displayed robust expression of DDR2 and immunohistochemical markers of lung adenocarcinoma comparable with previously generated models, though also displayed concomitant expression of the squamous cell markers p63 and SOX2. Tumor-derived cell lines were not solely DDR2 dependent and displayed upregulation of and partial dependence on MYCN. Combined treatment with the multitargeted DDR2 inhibitor dasatinib and BET inhibitor JQ1 inhibited tumor growth in vitro and in vivo. Together, these results suggest that DDR2 mutation can drive lung cancer initiation in vivo and provide a novel mouse model for lung cancer therapeutics studies.

Acquired resistance to dasatinib in lung cancer cell lines conferred by DDR2 gatekeeper mutation and NF1 loss.

The treatment of non-small cell lung cancer has evolved dramatically over the past decade with the adoption of widespread use of effective targeted therapies in patients with distinct molecular alterations. In lung squamous cell carcinoma (lung SqCC), recent studies have suggested that DDR2 mutations are a biomarker for therapeutic response to dasatinib and clinical trials are underway testing this hypothesis. Although targeted therapeutics are typically quite effective as initial therapy for patients with lung cancer, nearly all patients develop resistance with long-term exposure to targeted drugs. Here, we use DDR2-dependent lung cancer cell lines to model acquired resistance to dasatinib therapy. We perform targeted exome sequencing to identify two distinct mechanisms of acquired resistance: acquisition of the T654I gatekeeper mutation in DDR2 and loss of NF1. We show that NF1 loss activates a bypass pathway, which confers ERK dependency downstream of RAS activation. These results indicate that acquired resistance to dasatinib can occur via both second-site mutations in DDR2 and by activation of bypass pathways. These data may help to anticipate mechanisms of resistance that may be identified in upcoming clinical trials of anti-DDR2 therapy in lung cancer and suggest strategies to overcome resistance.

Inhibitor-sensitive FGFR2 and FGFR3 mutations in lung squamous cell carcinoma.

A comprehensive description of genomic alterations in lung squamous cell carcinoma (lung SCC) has recently been reported, enabling the identification of genomic events that contribute to the oncogenesis of this disease. In lung SCC, one of the most frequently altered receptor tyrosine kinase families is the fibroblast growth factor receptor (FGFR) family, with amplification or mutation observed in all four family members. Here, we describe the oncogenic nature of mutations observed in FGFR2 and FGFR3, each of which are observed in 3% of samples, for a mutation rate of 6% across both genes. Using cell culture and xenograft models, we show that several of these mutations drive cellular transformation. Transformation can be reversed by small-molecule FGFR inhibitors currently being developed for clinical use. We also show that mutations in the extracellular domains of FGFR2 lead to constitutive FGFR dimerization. In addition, we report a patient with an FGFR2-mutated oral SCC who responded to the multitargeted tyrosine kinase inhibitor pazopanib. These findings provide new insights into driving oncogenic events in a subset of lung squamous cancers, and recommend future clinical studies with FGFR inhibitors in patients with lung and head and neck SCC.

Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors.

UNLABELLED: The success in lung cancer therapy with programmed death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between EGF receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, CTL antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased CTLs and increased markers of T-cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T-cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape and mechanistically link treatment response to PD-1 inhibition. SIGNIFICANCE: We show that autochthonous EGFR-driven lung tumors inhibit antitumor immunity by activating the PD-1/PD-L1 pathway to suppress T-cell function and increase levels of proinflammatory cytokines. These findings indicate that EGFR functions as an oncogene through non-cell-autonomous mechanisms and raise the possibility that other oncogenes may drive immune escape.