Enhanced intratumoral delivery of immunomodulator MPLA via hyperbranched polyglycerol-coated biodegradable nanoparticles.

Immunomodulatory agents (IMA) have significant potential to enhance cancer treatment but have demonstrated limited efficacy beyond the preclinical setting due to poor pharmacokinetics and toxicity associated with systemic administration. On the other hand, when locally delivered, IMAs require repeated administration to optimize immune stimulation. To overcome these challenges, we encapsulated the toll-like receptor (TLR)4 agonist monophosphoryl lipid A (MPLA) within hyperbranched polyglycerol (HPG)-coated biodegradable nanoparticles (NP) engineered for gradual drug release from the nanoparticle core, resulting in a more persistent stimulation of anti-tumor immune responses while minimizing systemic side effects. In a model of malignant melanoma, we demonstrate that HPG-NP encapsulation significantly improves the antitumor efficacy of MPLA by enhancing its ability to remodel the tumor microenvironment (TME). Compared to free MPLA, HPG-NP-MPLA significantly increased the natural killer cell and cytotoxic T cell mediated antitumor immune response and tuned the tumor draining lymph nodes towards a T helper (Th)1 response. Furthermore, when combined with local delivery of a chemotherapeutic agent, HPG-NP-MPLA induces the conversion of an immunosuppressive TME to immunogenic TME and significantly improves survival.

Generation and optimization of off-the-shelf immunotherapeutics targeting TCR-Vß2+ T cell malignancy.

Current treatments for T cell malignancies encounter issues of disease relapse and off-target toxicity. Using T cell receptor (TCR)Vß2 as a model, here we demonstrate the rapid generation of an off-the-shelf allogeneic chimeric antigen receptor (CAR)-T platform targeting the clone-specific TCR Vß chain for malignant T cell killing while limiting normal cell destruction. Healthy donor T cells undergo CRISPR-induced TRAC, B2M and CIITA knockout to eliminate T cell-dependent graft-versus-host and host-versus-graft reactivity. Second generation 4-1BB/CD3zeta CAR containing high affinity humanized anti-Vß scFv is expressed efficiently on donor T cells via both lentivirus and adeno-associated virus transduction with limited detectable pre-existing immunoreactivity. Our optimized CAR-T cells demonstrate specific and persistent killing of Vß2+ Jurkat cells and Vß2+ patient derived malignant T cells, in vitro and in vivo, without affecting normal T cells. In parallel, we generate humanized anti-Vß2 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) by Fc-engineering for NK cell ADCC therapy.

Integrated transcriptome and trajectory analysis of cutaneous T-cell lymphoma identifies putative precancer populations.

The incidence of cutaneous T-cell lymphoma (CTCL) increases with age, and blood involvement portends a worse prognosis. To advance our understanding of the development of CTCL and identify potential therapeutic targets, we performed integrative analyses of paired single-cell RNA and T-cell receptor (TCR) sequencing of peripheral blood CD4+ T cells from patients with CTCL to reveal disease-unifying features. The malignant CD4+ T cells of CTCL showed highly diverse transcriptomic profiles across patients, with most displaying a mature Th2 differentiation and T-cell exhaustion phenotype. TCR-CDR3 peptide prediction analysis suggested limited diversity between CTCL samples, consistent with a role for a common antigenic stimulus. Potential of heat diffusion for affinity-based trajectory embedding transition analysis identified putative precancerous circulating populations characterized by an intermediate stage of gene expression and mutation level between the normal CD4+ T cells and malignant CTCL cells. We further revealed the therapeutic potential of targeting CD82 and JAK that endow the malignant CTCL cells with survival and proliferation advantages.

Chronic UV radiation-induced RORγt+ IL-22-producing lymphoid cells are associated with mutant KC clonal expansion.

Chronic ultraviolet (UV) radiation exposure is the greatest risk factor for cutaneous squamous cell carcinoma (cSCC) development, and compromised immunity accelerates this risk. Having previously identified that epidermal Langerhans cells (LC) facilitate the expansion of UV-induced mutant keratinocytes (KC), we sought to more fully elucidate the immune pathways critical to cutaneous carcinogenesis and to identify potential targets of intervention. Herein, we reveal that chronic UV induces and LC enhance a local immune shift toward RORγt+ interleukin (IL)-22/IL-17A-producing cells that occurs in the presence or absence of T cells while identifying a distinct RORγt+ Sca-1+ CD103+ ICOS+ CD2+/- CCR6+ intracellular CD3+ cutaneous innate lymphoid cell type-3 (ILC3) population (uvILC3) that is associated with UV-induced mutant KC growth. We further show that mutant KC clone size is markedly reduced in the absence of RORγt+ lymphocytes or IL-22, both observed in association with expanding KC clones, and find that topical application of a RORγ/γt inhibitor during chronic UV exposure reduces local expression of IL-22 and IL-17A while markedly limiting mutant p53 KC clonal expansion. We implicate upstream Toll-like receptor signaling in driving this immune response to chronic UV exposure, as MyD88/Trif double-deficient mice also show substantially reduced p53 island number and size. These data elucidate key immune components of chronic UV-induced cutaneous carcinogenesis that might represent targets for skin cancer prevention.

Nonsurgical treatment of skin cancer with local delivery of bioadhesive nanoparticles.

Keratinocyte-derived carcinomas, including squamous cell carcinoma (SCC), comprise the most common malignancies. Surgical excision is the therapeutic standard but is not always clinically feasible, and currently available alternatives are limited to superficial tumors. To address the need for a nonsurgical treatment for nodular skin cancers like SCC, we developed a bioadhesive nanoparticle (BNP) drug delivery system composed of biodegradable polymer, poly(lactic acid)-hyperbranched polyglycerol (PLA-HPG), encapsulating camptothecin (CPT). Nanoparticles (NPs) of PLA-HPG are nonadhesive NPs (NNPs), which are stealthy in their native state, but we have previously shown that conversion of the vicinal diols of HPG to aldehydes conferred NPs the ability to form strong covalent bonds with amine-rich surfaces. Herein, we show that these BNPs have significantly enhanced binding to SCC tumor cell surfaces and matrix proteins, thereby significantly enhancing the therapeutic efficacy of intratumoral drug delivery. Tumor injection of BNP-CPT resulted in tumor retention of CPT at ∼50% at 10 d postinjection, while CPT was undetectable in NNP-CPT or free (intralipid) CPT-injected tumors at that time. BNP-CPT also significantly reduced tumor burden, with a portion (∼20%) of BNP-CPT-treated established tumors showing histologic cure. Larger, more fully established PDV SCC tumors treated with a combination of BNP-CPT and immunostimulating CpG oligodeoxynucleotides exhibited enhanced survival relative to controls, revealing the potential for BNP delivery to be used along with local tumor immunotherapy. Taken together, these results indicate that percutaneous delivery of a chemotherapeutic agent via BNPs, with or without adjuvant immunostimulation, represents a viable, nonsurgical alternative for treating cutaneous malignancy.

Research Techniques Made Simple: Preclinical Development of Combination Antitumor Targeted Therapies in Dermatology.

The identification and application of targeted therapies that inhibit critical pathways in malignant cells have shown tremendous promise for improving clinical outcomes for patients with advanced cutaneous malignancies. However, tumor cell heterogeneity, development of drug resistance, and risks of off-target effects remain barriers to prolonged remission and definitive cure. Herein, we describe the potential that combinations of antitumor targeted agents may offer in overcoming these challenges and detail techniques whereby promising combination regimens can be identified and further evaluated preclinically. Cancer cell lines and primary patient-derived malignant cells can be utilized to perform dose-response screenings in vitro for individual targeted agents before moving toward the evaluation of potential synergistic combinations. Mathematical analyses, including the Chou-Talalay method, determine combination indices and Hill slopes that permit relative comparisons among various drug combinations by quantification of synergistic activities. Further preclinical in vivo evaluation of promising single versus combination regimens may be studied in relevant mouse models of cutaneous malignancy. Ultimately, the formulation of combination targeted therapy regimens may be more broadly effective and less toxic, helping to better inform clinical trial design and prioritization.

JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL.

Cutaneous T-cell lymphoma (CTCL) is a malignancy of skin-homing T lymphocytes that is more likely to involve the peripheral blood in advanced stages. For such patients with advanced disease, there are few available systemic treatment options, and prognosis remains poor. Exome sequencing studies of CTCL have suggested therapeutic targets, including within the JAK/STAT pathway, but JAK inhibition strategies may be limited by patient-specific mutational status. Because our recent research has highlighted the potential roles of single and combination approaches specifically using BCL2, bromodomain and extra-terminal domain (BET), and histone deacetylase (HDAC) inhibition, we aimed to investigate the effects of JAK inhibition on CTCL cells and established CTCL cell lines when paired with these and other targeting agents. Peripheral blood malignant CTCL isolates exhibited differential responses to JAK inhibition, with JAK2 expression levels negatively correlating to 50% inhibitory concentration (IC50) values. Regardless of single-agent sensitivity, JAK inhibition potentiated malignant cell cytotoxicity in combination with BCL2, BET, HDAC, or proteasome inhibition. Combination inhibition of JAK and BCL2 showed the strongest potentiation of CTCL cytotoxicity, driven by both intrinsic and extrinsic apoptosis pathways. JAK inhibition decreased expression of BCL2 in the high-responder samples, suggesting a putative mechanism for this combination activity. These results indicate that JAK inhibition may have major effects on CTCL cells, and that combination strategies using JAK inhibition may allow for more generalized cytotoxic effects against the malignant cells from patients with CTCL. Such preclinical assessments help inform prioritization for combination targeted drug approaches for clinical utilization in the treatment of CTCL.

BET inhibition in advanced cutaneous T cell lymphoma is synergistically potentiated by BCL2 inhibition or HDAC inhibition.

While several systemic therapies are approved for cutaneous T cell lymphoma (CTCL), a non-Hodgkin lymphoma of skin-homing T cells that may involve lymph nodes and peripheral blood in advanced stages, relapses are common. Mutational analysis of CTCL cells has revealed frequent amplification of the MYC oncogene, and bromodomain and extraterminal (BET) protein inhibitors have been shown to repress MYC expression in various malignancies. Towards a potential novel therapy, we thus sought to examine the effect of BET inhibition on CTCL cells in vitro. Each of the four tested BET inhibitors (JQ1, ABBV-075, I-BET762, CPI-0610) consistently induced dose-dependent decreases in viability of isolated patient-derived CTCL cells and established CTCL cell lines (MyLa, Sez4, HH, Hut78). This effect was synergistically potentiated by combination of BET inhibition with BCL2 inhibition (e.g. venetoclax) or histone deacetylase (HDAC) inhibition (e.g. vorinostat or romidepsin). There was also a marked increase in caspase 3/7 activation when JQ1 was combined with either vorinostat or romidepsin, confirming that the observed synergies are due in major part to induction of apoptosis. Furthermore, MYC and BCL2 expression were each synergistically repressed when CTCL cells were treated with JQ1 plus HDAC inhibitors, suggesting cooperative activities at the level of epigenetic regulation. Taken together, these data indicate that targeting BET proteins in CTCL represents a promising novel therapeutic strategy that may be substantially potentiated by combination with BCL2 or HDAC inhibition.

Synergy of BCL2 and histone deacetylase inhibition against leukemic cells from cutaneous T-cell lymphoma patients.

The presence and degree of peripheral blood involvement in patients with cutaneous T-cell lymphoma (CTCL) portend a worse clinical outcome. Available systemic therapies for CTCL may variably decrease tumor burden and improve quality of life, but offer limited effects on survival; thus, novel approaches to the treatment of advanced stages of this non-Hodgkin lymphoma are clearly warranted. Mutational analyses of CTCL patient peripheral blood malignant cell samples suggested the antiapoptotic mediator B-cell lymphoma 2 (BCL2) as a potential therapeutic target. To test this, we developed a screening assay for evaluating the sensitivity of CTCL cells to targeted molecular agents, and compared a novel BCL2 inhibitor, venetoclax, alone and in combination with a histone deacetylase (HDAC) inhibitor, vorinostat or romidepsin. Peripheral blood CTCL malignant cells were isolated from 25 patients and exposed ex vivo to the 3 drugs alone and in combination, and comparisons were made to 4 CTCL cell lines (Hut78, Sez4, HH, MyLa). The majority of CTCL patient samples were sensitive to venetoclax, and BCL2 expression levels were negatively correlated (r = -0.52; P =018) to 50% inhibitory concentration values. Furthermore, this anti-BCL2 effect was markedly potentiated by concurrent HDAC inhibition with 93% of samples treated with venetoclax and vorinostat and 73% of samples treated with venetoclax and romidepsin showing synergistic effects. These data strongly suggest that concurrent BCL2 and HDAC inhibition may offer synergy in the treatment of patients with advanced CTCL. By using combination therapies and correlating response to gene expression in this way, we hope to achieve more effective and personalized treatments for CTCL.

Genomic landscape of cutaneous T cell lymphoma.

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma of skin-homing T lymphocytes. We performed exome and whole-genome DNA sequencing and RNA sequencing on purified CTCL and matched normal cells. The results implicate mutations in 17 genes in CTCL pathogenesis, including genes involved in T cell activation and apoptosis, NF-κB signaling, chromatin remodeling and DNA damage response. CTCL is distinctive in that somatic copy number variants (SCNVs) comprise 92% of all driver mutations (mean of 11.8 pathogenic SCNVs versus 1.0 somatic single-nucleotide variant per CTCL). These findings have implications for new therapeutics.

Langerhans Cells Facilitate UVB-Induced Epidermal Carcinogenesis.

UVB light is considered the major environmental inducer of human keratinocyte (KC) DNA mutations, including within the tumor-suppressor gene p53, and chronic exposure is associated with cutaneous squamous cell carcinoma formation. Langerhans cells (LCs) comprise a dendritic network within the suprabasilar epidermis, yet the role of LCs in UVB-induced carcinogenesis is largely unknown. Herein we show that LC-intact epidermis develops UVB-induced tumors more readily than LC-deficient epidermis. Although levels of epidermal cyclopyrimidine dimers following acute UVB exposure are equivalent in the presence or absence of LCs, chronic UVB-induced p53 mutant clonal islands expand more readily in association with LCs, which remain largely intact and are preferentially found in proximity to the expanding mutant KC populations. The observed LC facilitation of mutant p53 clonal expansion is completely αß and γδ T-cell independent and is associated with increased intraepidermal expression of IL-22 and the presence of group 3 innate lymphoid cells. These data demonstrate that LCs have a key role in UVB-induced cutaneous carcinogenesis and suggest that LCs locally stimulate KC proliferation and innate immune cells that provoke tumor outgrowth.

Mechanisms of chemical cooperative carcinogenesis by epidermal Langerhans cells.

Cutaneous squamous cell carcinoma (SCC) is the most prevalent invasive malignancy with metastatic potential. The epidermis is exposed to a variety of environmental DNA-damaging chemicals, principal among which are polyaromatic hydrocarbons (PAHs) ubiquitous in the environment, tobacco smoke, and broiled meats. Langerhans cells (LCs) comprise a network of dendritic cells situated adjacent to basal, suprabasal, and follicular infundibular keratinocytes that when mutated can give rise to SCC, and LC-intact mice are markedly more susceptible than LC-deficient mice to chemical carcinogenesis provoked by initiation with the model PAH, 7,12-dimethylbenz[a]anthracene (DMBA). LCs rapidly internalize and accumulate DMBA as numerous membrane-independent cytoplasmic foci. Repopulation of LC-deficient mice using fetal liver LC-precursors restores DMBA-induced tumor susceptibility. LC expression of p450 enzyme CYP1B1 is required for maximal rapid induction of DNA-damage within adjacent keratinocytes and their efficient neoplastic transformation; however, effects of tumor progression also attributable to the presence of LC were revealed as CYP1B1 independent. Thus, LCs make multifaceted contributions to cutaneous carcinogenesis, including via the handling and metabolism of chemical mutagens. Such findings suggest a cooperative carcinogenesis role for myeloid-derived cells resident within cancer susceptible epithelial tissues principally by influencing early events in malignant transformation.

Characterization of the DNA copy-number genome in the blood of cutaneous T-cell lymphoma patients.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous non-Hodgkin's lymphoma that may variably involve the skin, lymph nodes, and peripheral blood. Malignant burden ranges from cutaneous patches and plaques with little evidence of blood involvement to erythroderma often in association with frank leukemia, as in Sézary syndrome. Toward a better understanding of the pathogenesis of this CD4+ T-cell malignancy, we conducted a high-resolution genomic analysis combining DNA (23 samples) and mRNA (12 samples) data of peripheral blood isolates from CTCL patients across a spectrum of stages. Strikingly, even patients with limited involvement, e.g., normal CD4 counts, contained significant copy-number alterations. Defining genomic characteristics of CTCL blood involvement included gains on 8q and 17q, and deletions on 17p and chromosome 10. A consensus analysis of 108 leukemic CTCL samples demonstrated global similarities among patients with varied blood involvement, narrowing 38 of 62 loci. Toward an annotated framework for in vitro testing, we also characterized genomic alterations in five CTCL cell lines (HH, HUT78, PNO, SeAx, and Sez4), revealing intact core features of leukemic CTCL. Together, these studies produce the most comprehensive view of the leukemic CTCL genome to date, with implications for pathogenesis, molecular classification, and potential future therapeutic developments.

Skint-1 is a highly specific, unique selecting component for epidermal T cells.

αß T-cell repertoire selection is mediated by peptide-MHC complexes presented by thymic epithelial or myeloid cells, and by lipid-CD1 complexes expressed by thymocytes. γδ T-cell repertoire selection, by contrast, is largely unresolved. Mice mutant for Skint-1, a unique Ig superfamily gene, do not develop canonical Vγ5Vδ1(+) dendritic epidermal T cells. This study shows that transgenic Skint-1, across a broad range of expression levels, precisely and selectively determines the Vγ5Vδ1(+) dendritic epidermal T-cell compartment. Skint-1 is expressed by medullary thymic epithelial cells, and unlike lipid-CD1 complexes, must be expressed by stromal cells to function efficiently. Its unusual transmembrane-cytoplasmic regions severely limit cell surface expression, yet increasing this or, conversely, retaining Skint1 intracellularly markedly compromises function. Each Skint1 domain appears nonredundant, including a unique decamer specifying IgV-domain processing. This investigation of Skint-1 biology points to complex events underpinning the positive selection of an intraepithelial γδ repertoire.