Investigations on keloid pathogenesis and therapy

Ph.DDOCTOR OF PHILOSOPH

The Post-Apoptotic Fate of RNAs Identified Through High-Throughput Sequencing of Human Hair

The hair of all mammals consists of terminally differentiated cells that undergo a specialized form of apoptosis called cornification. While DNA is destroyed during cornification, the extent to which RNA is lost is unknown. Here we find that multiple types of RNA are incompletely degraded after hair shaft formation in both mouse and human. Notably, mRNAs and short regulatory microRNAs (miRNAs) are stable in the hair as far as 10 cm from the scalp. To better characterize the post-apoptotic RNAs that escape degradation in the hair, we performed sequencing (RNA-seq) on RNA isolated from hair shafts pooled from several individuals. This hair shaft RNA library, which encompasses different hair types, genders, and populations, revealed 7,193 mRNAs, 449 miRNAs and thousands of unannotated transcripts that remain in the post-apoptotic hair. A comparison of the hair shaft RNA library to that of viable keratinocytes revealed surprisingly similar patterns of gene coverage and indicates that degradation of RNA is highly inefficient during apoptosis of hair lineages. The generation of a hair shaft RNA library could be used as months of accumulated transcriptional history useful for retrospective detection of disease, drug response and environmental exposure

Activated Kras Alters Epidermal Homeostasis of Mouse Skin, Resulting in Redundant Skin and Defective Hair Cycling

Germline mutations in the RAS–mitogen-activated protein kinase (RAS/MAPK) pathway are associated with genodermatoses, characterized by cutaneous, cardiac, and craniofacial defects, and cancer predisposition. Whereas activating mutations in HRAS are associated with the vast majority of patients with Costello syndrome, mutations in its paralog, KRAS, are rare. To better understand the disparity among RAS paralogs in human syndromes, we generated mice that activate a gain-of-function Kras allele (Lox-Stop-Lox (LSL)-KrasG12D) in ectodermal tissue using two different Cre transgenic lines. Using Msx2-Cre or ligand-inducible keratin 15 (K15)-CrePR, the embryonic effects of activated Kras were bypassed and the effects of KrasG12D expression from its endogenous promoter were determined. We found that KrasG12D induced redundant skin, papillomas, shortened nails, and hair loss. Redundant skin was associated with basal keratinocyte hyperplasia and an increase in body surface area. Paradoxically, KrasG12D also prevented hair cycle activation. We find that KrasG12D blocks proliferation in the bulge region of the hair follicle, when activated through Msx2-Cre but not through K15-CrePR. These studies reveal that KRAS, although infrequently involved in RAS/MAPK syndromes, is capable of inducing multiple cutaneous features that grossly resemble human RAS/MAPK syndromes

Sox2 Cooperates with Lkb1 Loss in a Mouse Model of Squamous Cell Lung Cancer

Squamous cell carcinoma (SCC) of the lung is the second most common subtype of lung cancer. With limited treatment options, the 5-year survival rate of SCC is only 15%. Although genomic alterations in SCC have been characterized, identifying the alterations that drive SCC is critical for improving treatment strategies. Mouse models of SCC are currently limited. Using lentiviral delivery of Sox2 specifically to the mouse lung, we tested the ability of Sox2 to promote tumorigenesis in multiple tumor suppressor backgrounds. Expression of Sox2, frequently amplified in human SCC, specifically cooperates with loss of Lkb1 to promote squamous lung tumors. Mouse tumors exhibit characteristic histopathology and biomarker expression similar to human SCC. They also mimic human SCCs by activation of therapeutically relevant pathways including STAT and mTOR. This model may be utilized to test the contribution of additional driver alterations in SCC, as well as for preclinical drug discovery

Intratumoral Electroporation of Plasmid Encoded IL12 and Membrane-Anchored Anti-CD3 Increases Systemic Tumor Immunity.

Intratumoral delivery of plasmid IL12 via electroporation (IT-tavo-EP) induces localized expression of IL12 leading to regression of treated and distant tumors with durable responses and minimal toxicity. A key driver in amplifying this local therapy into a systemic response is the magnitude and composition of immune infiltrate in the treated tumor. While intratumoral IL12 typically increases the density of CD3+ tumor-infiltrating lymphocytes (TIL), this infiltrate is composed of a broad range of T-cell subsets, including activated tumor-specific T cells, less functional bystander T cells, as well as suppressive T regulatory cells. To encourage a more favorable on-treatment tumor microenvironment (TME), we explored combining this IL12 therapy with an intratumoral polyclonal T-cell stimulator membrane-anchored anti-CD3 to productively engage a diverse subset of lymphocytes including the nonreactive and suppressive T cells. This study highlighted that combined intratumoral electroporation of IL12 and membrane-anchored anti-CD3 plasmids can enhance cytokine production, T-cell cytotoxicity, and proliferation while limiting the suppressive capacity within the TME. These collective antitumor effects not only improve regression of treated tumors but drive systemic immunity with control of nontreated contralateral tumors in vivo. Moreover, combination of IL12 and anti-CD3 restored the function of TIL isolated from a patient with melanoma actively progressing on programmed cell death protein 1 (PD-1) checkpoint inhibitor therapy. IMPLICATIONS: This DNA-encodable polyclonal T-cell stimulator (membrane-anchored anti-CD3 plasmid) may represent a key addition to intratumoral IL12 therapies in the clinic

Amplification of the CXCR3/CXCL9 axis via intratumoral electroporation of plasmid CXCL9 synergizes with plasmid IL-12 therapy to elicit robust anti-tumor immunity.

Clinical studies have demonstrated that local expression of the cytokine IL-12 drives interferon-gamma expression and recruits T cells to the tumor microenvironment, ultimately yielding durable systemic T cell responses. Interrogation of longitudinal biomarker data from our late-stage melanoma trials identified a significant on-treatment increase of intratumoral CXCR3 transcripts that was restricted to responding patients, underscoring the clinical relevance of tumor-infiltrating CXCR3+ immune cells. In this study, we sought to understand if the addition of DNA-encodable CXCL9 could augment the anti-tumor immune responses driven by intratumoral IL-12. We show that localized IL-12 and CXCL9 treatment reshapes the tumor microenvironment to promote dendritic cell licensing and CD8+ T cell activation. Additionally, this combination treatment results in a significant abscopal anti-tumor response and provides a concomitant benefit to anti-PD-1 therapies. Collectively, these data demonstrate that a functional tumoral CXCR3/CXCL9 axis is critical for IL-12 anti-tumor efficacy. Furthermore, restoring or amplifying the CXCL9 gradient in the tumors via intratumoral electroporation of plasmid CXCL9 can not only result in efficient trafficking of cytotoxic CD8+ T cells into the tumor but can also reshape the microenvironment to promote systemic immune response

The Lineage-Defining Transcription Factors SOX2 and NKX2-1 Determine Lung Cancer Cell Fate and Shape the Tumor Immune Microenvironment

The major types of non-small-cell lung cancer (NSCLC)-squamous cell carcinoma and adenocarcinoma-have distinct immune microenvironments. We developed a genetic model of squamous NSCLC on the basis of overexpression of the transcription factor Sox2, which specifies lung basal cell fate, and loss of the tumor suppressor Lkb1 (SL mice). SL tumors recapitulated gene-expression and immune-infiltrate features of human squamous NSCLC; such features included enrichment of tumor-associated neutrophils (TANs) and decreased expression of NKX2-1, a transcriptional regulator that specifies alveolar cell fate. In Kras-driven adenocarcinomas, mis-expression of Sox2 or loss of Nkx2-1 led to TAN recruitment. TAN recruitment involved SOX2-mediated production of the chemokine CXCL5. Deletion of Nkx2-1 in SL mice (SNL) revealed that NKX2-1 suppresses SOX2-driven squamous tumorigenesis by repressing adeno-to-squamous transdifferentiation. Depletion of TANs in SNL mice reduced squamous tumors, suggesting that TANs foster squamous cell fate. Thus, lineage-defining transcription factors determine the tumor immune microenvironment, which in turn might impact the nature of the tumor

MYC Drives Progression of Small Cell Lung Cancer to a Variant Neuroendocrine Subtype with Vulnerability to Aurora Kinase Inhibition

Loss of the tumor suppressors RB1 and TP53 and MYC amplification are frequent oncogenic events in small cell lung cancer (SCLC). We show that Myc expression cooperates with Rb1 and Trp53 loss in the mouse lung to promote aggressive, highly metastatic tumors, that are initially sensitive to chemotherapy followed by relapse, similar to human SCLC. Importantly, MYC drives a neuroendocrine-low variant subset of SCLC with high NEUROD1 expression corresponding to transcriptional profiles of human SCLC. Targeted drug screening reveals that SCLC with high MYC expression is vulnerable to Aurora kinase inhibition, which, combined with chemotherapy, strongly suppresses tumor progression and increases survival. These data identify molecular features for patient stratification and uncover a potential targeted treatment approach for MYC-driven SCLC