NSD2 dimethylation at H3K36 promotes lung adenocarcinoma pathogenesis.

The etiological role of NSD2 enzymatic activity in solid tumors is unclear. Here we show that NSD2, via H3K36me2 catalysis, cooperates with oncogenic KRAS signaling to drive lung adenocarcinoma (LUAD) pathogenesis. In vivo expression of NSD2E1099K, a hyperactive variant detected in individuals with LUAD, rapidly accelerates malignant tumor progression while decreasing survival in KRAS-driven LUAD mouse models. Pathologic H3K36me2 generation by NSD2 amplifies transcriptional output of KRAS and several complementary oncogenic gene expression programs. We establish a versatile in vivo CRISPRi-based system to test gene functions in LUAD and find that NSD2 loss strongly attenuates tumor progression. NSD2 knockdown also blocks neoplastic growth of PDXs (patient-dervived xenografts) from primary LUAD. Finally, a treatment regimen combining NSD2 depletion with MEK1/2 inhibition causes nearly complete regression of LUAD tumors. Our work identifies NSD2 as a bona fide LUAD therapeutic target and suggests a pivotal epigenetic role of the NSD2-H3K36me2 axis in sustaining oncogenic signaling.

Solution Equilibrium Study of Divalent Metal Ions with Phenylpropanoid Derivatives and Acetylcysteine Ligands.

Solution equilibrium of divalent metal ions (M=Mn2+, Co2+, Ni2+, Cu2+ and Zn2+) with caffeic acid (ligand C) or dihydrocaffeic acid (ligand D) in binary system, and with acetylcysteine (ligand N) in ternary system were investigated at condition similar to human physiological temperature of 310.15 K and ionic strength of 0.15 mol·dm-3 NaCl. Potentiometry technique was used for the determination of formation constant (log ß) assisted by spectrophotometry technique. The results indicated the formation of [ML], [MLH], [ML2], [ML2H] in binary species and [MLN], [MNLH], [MNLH2] in ternary species, where L represents ligands C or D. It was found that ligand D formed more stable complexes than that of ligand C, which were affected by the presence of double bond in the carboxylate moiety of ligand C. The speciation diagrams were simulated by HySS and discussed briefly, additionally the tendency of ternary complexes was evaluated from parameters Δ log KM and log X.

The MADS box transcription factor MEF2C regulates melanocyte development and is a direct transcriptional target and partner of SOX10.

Waardenburg syndromes are characterized by pigmentation and autosensory hearing defects, and mutations in genes encoding transcription factors that control neural crest specification and differentiation are often associated with Waardenburg and related disorders. For example, mutations in SOX10 result in a severe form of Waardenburg syndrome, Type IV, also known as Waardenburg-Hirschsprung disease, characterized by pigmentation and other neural crest defects, including defective innervation of the gut. SOX10 controls neural crest development through interactions with other transcription factors. The MADS box transcription factor MEF2C is an important regulator of brain, skeleton, lymphocyte and cardiovascular development and is required in the neural crest for craniofacial development. Here, we establish a novel role for MEF2C in melanocyte development. Inactivation of Mef2c in the neural crest of mice results in reduced expression of melanocyte genes during development and a significant loss of pigmentation at birth due to defective differentiation and reduced abundance of melanocytes. We identify a transcriptional enhancer of Mef2c that directs expression to the neural crest and its derivatives, including melanocytes, in transgenic mouse embryos. This novel Mef2c neural crest enhancer contains three functional SOX binding sites and a single essential MEF2 site. We demonstrate that Mef2c is a direct transcriptional target of SOX10 and MEF2 via this evolutionarily conserved enhancer. Furthermore, we show that SOX10 and MEF2C physically interact and function cooperatively to activate the Mef2c gene in a feed-forward transcriptional circuit, suggesting that MEF2C might serve as a potentiator of the transcriptional pathways affected in Waardenburg syndromes.

Targeting DNA Damage Response Promotes Antitumor Immunity through STING-Mediated T-cell Activation in Small Cell Lung Cancer.

Despite recent advances in the use of immunotherapy, only a minority of patients with small cell lung cancer (SCLC) respond to immune checkpoint blockade (ICB). Here, we show that targeting the DNA damage response (DDR) proteins PARP and checkpoint kinase 1 (CHK1) significantly increased protein and surface expression of PD-L1. PARP or CHK1 inhibition remarkably potentiated the antitumor effect of PD-L1 blockade and augmented cytotoxic T-cell infiltration in multiple immunocompetent SCLC in vivo models. CD8+ T-cell depletion reversed the antitumor effect, demonstrating the role of CD8+ T cells in combined DDR-PD-L1 blockade in SCLC. We further demonstrate that DDR inhibition activated the STING/TBK1/IRF3 innate immune pathway, leading to increased levels of chemokines such as CXCL10 and CCL5 that induced activation and function of cytotoxic T lymphocytes. Knockdown of cGAS and STING successfully reversed the antitumor effect of combined inhibition of DDR and PD-L1. Our results define previously unrecognized innate immune pathway-mediated immunomodulatory functions of DDR proteins and provide a rationale for combining PARP/CHK1 inhibitors and immunotherapies in SCLC. SIGNIFICANCE: Our results define previously unrecognized immunomodulatory functions of DDR inhibitors and suggest that adding PARP or CHK1 inhibitors to ICB may enhance treatment efficacy in patients with SCLC. Furthermore, our study supports a role of innate immune STING pathway in DDR-mediated antitumor immunity in SCLC.See related commentary by Hiatt and MacPherson, p. 584.This article is highlighted in the In This Issue feature, p. 565.

Chemosensitive Relapse in Small Cell Lung Cancer Proceeds through an EZH2-SLFN11 Axis.

Small cell lung cancer is initially highly responsive to cisplatin and etoposide but in almost every case becomes rapidly chemoresistant, leading to death within 1 year. We modeled acquired chemoresistance in vivo using a series of patient-derived xenografts to generate paired chemosensitive and chemoresistant cancers. Multiple chemoresistant models demonstrated suppression of SLFN11, a factor implicated in DNA-damage repair deficiency. In vivo silencing of SLFN11 was associated with marked deposition of H3K27me3, a histone modification placed by EZH2, within the gene body of SLFN11, inducing local chromatin condensation and gene silencing. Inclusion of an EZH2 inhibitor with standard cytotoxic therapies prevented emergence of acquired resistance and augmented chemotherapeutic efficacy in both chemosensitive and chemoresistant models of small cell lung cancer.

Loss of Pten Disrupts the Thymic Epithelium and Alters Thymic Function.

The thymus is the site of T cell development and selection. In addition to lymphocytes, the thymus is composed of several types of stromal cells that are exquisitely organized to create the appropriate environment and microenvironment to support the development and selection of maturing T cells. Thymic epithelial cells (TECs) are one of the more important cell types in the thymic stroma, and they play a critical role in selecting functional T cell clones and supporting their development. In this study, we used a mouse genetics approach to investigate the consequences of deleting the Pten tumor suppressor gene in the TEC compartment of the developing thymus. We found that PTEN deficiency in TECs results in a smaller thymus with significantly disordered architecture and histology. Accordingly, loss of PTEN function also results in decreased T cells with a shift in the distribution of T cell subtypes towards CD8+ T cells. These experiments demonstrate that PTEN is critically required for the development of a functional thymic epithelium in mice. This work may help better understand the effects that certain medical conditions or clinical interventions have upon the thymus and immune function.

Identification and Targeting of Long-Term Tumor-Propagating Cells in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a neuroendocrine lung cancer characterized by fast growth, early dissemination, and rapid resistance to chemotherapy. We identified a population of long-term tumor-propagating cells (TPCs) in a mouse model of SCLC. This population, marked by high levels of EpCAM and CD24, is also prevalent in human primary SCLC tumors. Murine SCLC TPCs are numerous and highly proliferative but not intrinsically chemoresistant, indicating that not all clinical features of SCLC are linked to TPCs. SCLC TPCs possess a distinct transcriptional profile compared to non-TPCs, including elevated MYC activity. Genetic and pharmacological inhibition of MYC in SCLC cells to non-TPC levels inhibits long-term propagation but not short-term growth. These studies identify a highly tumorigenic population of SCLC cells in mouse models, cell lines, and patient tumors and a means to target them in this most fatal form of lung cancer.

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer.

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

Characterization of melanosomes in murine Hermansky-Pudlak syndrome: mechanisms of hypopigmentation.

The Hermansky-Pudlak syndrome is a genetically heterogeneous autosomal recessive disorder affecting mice and humans, which causes oculocutaneous albinism, prolonged bleeding, and in some cases, pulmonary fibrosis or granulomatous colitis. We previously demonstrated that the gene defects causing murine Hermansky-Pudlak syndrome cause blocks in melanosome biogenesis and/or trafficking in 10 Hermansky-Pudlak syndrome strains. Here, we report an in vivo quantitative analysis on five additional murine models of the Hermansky-Pudlak syndrome. We demonstrate that all strains examined here except for ashen have defects in morphogenesis, the most severely affected is sandy, muted, and buff followed by subtle gray. The ashen strain only has a defect in secretion, as indicated by retention of melanosomes in melanocytes. We document three cellular mechanisms contributing to the hypopigmentation seen in the Hermansky-Pudlak syndrome: (1) exocytosis of immature hypopigmented melanosomes from melanocytes with subsequent keratinocyte uptake; (2) decreased intramelanocyte steady-state numbers of melanosomes available for transfer to keratinocytes; and (3) accumulation of melanosomes within melanocytes due to defective exocytosis, as seen in ashen. We also report that melanosomes in the DBA/2J strain, the parental strain of the Hermansky-Pudlak syndrome strain sandy, are abnormal, indicating that aberrant biogenesis of melanosomes may play a part in the pathogenesis of pigmentary glaucoma observed in these mice.

Melanosome morphologies in murine models of hermansky-pudlak syndrome reflect blocks in organelle development.

Hermansky-Pudlak syndrome is an autosomal recessive disease characterized by pigment dilution and prolonged bleeding time. At least 15 mutant mouse strains have been classified as models of Hermansky-Pudlak syndrome. Some of the genes are implicated in intracellular vesicle trafficking: budding, targeting, and secretion. Many of the Hermansky-Pudlak syndrome genes remain uncharacterized and their functions are unknown. Clues to the functions of these genes can be found by analyzing the physiologic and cellular phenotypes. Here we have examined the morphology of the melanosomes in the skin of 10 of the mutant mouse Hermansky-Pudlak syndrome strains by transmission electron microscopy. We demonstrate that the morphologies reflect inhibition of organelle maturation or transfer. The Hermansky-Pudlak syndrome strains are classified into morphologic groups characterized by the step at which melanosome biogenesis or transfer to keratinocytes is inhibited, with the cappuccino strain observed to be blocked at the earliest step and gunmetal blocked at the latest step. We show that all Hermansky-Pudlak syndrome mutant strains except gunmetal have an increase in unpigmented or hypopigmented immature melanosomal forms, leading to the hypopigmented coat colors seen in these strains. In contrast, the hypopigmentation seen in the gunmetal strain is due to the retention of melanosomes in melanocytes, and inefficient transfer into keratinocytes.

Multidrug resistance decreases with mutations of melanosomal regulatory genes.

Whereas resistance to chemotherapy has long impeded effective treatment of metastatic melanoma, the mechanistic basis of this resistance remains unknown. One possible mechanism of drug resistance is alteration of intracellular drug distribution either by drug efflux or sequestration into intracellular organelles. Melanomas, as well as primary melanocytes from which they arise, have intracellular organelles, called melanosomes, wherein the synthesis and storage of the pigment melanin takes place. In this study, comparisons of congenic cells with and without functional molecules regulating melanosome formation show that sensitivity to the chemotherapeutic agent cis-diaminedichloroplatinum II (cis-platin) significantly increases with the mutation of genes regulating melanosome formation, concomitant disruption of melanosome morphology, and loss of mature melanosomes. Absence of the melanosomal structural protein gp100/Pmel17 causes increased cis-platin sensitivity. Independent mutations in three separate genes that regulate melanosome biogenesis (Dtnbp1, Pldn, Vps33a) also result in increased cis-platin sensitivity. In addition, a mutation of the gene encoding the integral melanosomal protein tyrosinase, resulting in aberrant melanosome formation, also causes increased cis-platin sensitivity. Furthermore, sensitivity to agents in other chemotherapeutic classes (e.g., vinblastine and etoposide) also increased with the mutation of Pldn. In contrast, a mutation in another melanosomal regulatory gene, Hps1, minimally affects melanosome biogenesis, preserves the formation of mature melanosomes, and has no effect on cis-platin or vinblastine response. Together, these data provide the first direct evidence that melanosomal regulatory genes influence drug sensitivity and that the presence of mature melanosomes likely contributes to melanoma resistance to therapy.

Hermansky-Pudlak HPS1/pale ear gene regulates epidermal and dermal melanocyte development.

The pale ear (ep) mouse strain is a model for the Hermansky-Pudlak syndrome type 1 (HPS-1), an autosomal recessive disorder causing pigmentary dilution, visual disturbances, bleeding diatheses, pulmonary fibrosis, and granulomatous colitis. The ep mice have a coat color very similar to the black-colored parental strain, C57BL/6. However, the ears and tails of ep mice are significantly hypopigmented compared with the control animals, suggesting that the gene mutation in ep mice reveals a differential regulation of melanocyte function in dorsal back skin melanocytes versus tail or ear skin. In this study, we analyzed the mutant phenotype in detail and determined that in the tail, the defective gene causes delayed onset of interfollicular epidermal melanocyte tyrosinase activity, decreased numbers of melanocytes in the interfollicular epidermis and dermis, and severe immaturity of tail epidermal melanosomes, findings not observed in dorsal back follicular melanocytes. These results highlight differences between follicular and interfollicular melanocyte biology and demonstrate that defects in the ep protein not only affect melanosome biogenesis, but also play a developmental role in determining interfollicular epidermal and dermal melanocyte function. The implications of these findings for the mechanisms governing physiologic variation in human pigmentation and for the pathogenesis of vitiligo are discussed.