Targeting intracellular oncoproteins with dimeric IgA promotes expulsion from the cytoplasm and immune-mediated control of epithelial cancers.

Dimeric IgA (dIgA) can move through cells via the IgA/IgM polymeric immunoglobulin receptor (PIGR), which is expressed mainly on mucosal epithelia. Here, we studied the ability of dIgA to target commonly mutated cytoplasmic oncodrivers. Mutation-specific dIgA, but not IgG, neutralized KRASG12D within ovarian carcinoma cells and expelled this oncodriver from tumor cells. dIgA binding changed endosomal trafficking of KRASG12D from accumulation in recycling endosomes to aggregation in the early/late endosomes through which dIgA transcytoses. dIgA targeting of KRASG12D abrogated tumor cell proliferation in cell culture assays. In vivo, KRASG12D-specific dIgA1 limited the growth of KRASG12D-mutated ovarian and lung carcinomas in a manner dependent on CD8+ T cells. dIgA specific for IDH1R132H reduced colon cancer growth, demonstrating effective targeting of a cytoplasmic oncodriver not associated with surface receptors. dIgA targeting of KRASG12D restricted tumor growth more effectively than small-molecule KRASG12D inhibitors, supporting the potential of this approach for the treatment of human cancers.

Sézary syndrome originates from heavily mutated hematopoietic progenitors.

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains unclear. Using single-cell RNA or T-cell receptor (TCR) sequencing of 32 619 CD3+CD4+ and CD26+/CD7+ and 29 932 CD3+CD4+ and CD26-/CD7- lymphocytes from the peripheral blood of 7 patients with CTCL, coupled to single-cell ATAC-sequencing of 26,411 CD3+CD4+ and CD26+/CD7+ and 33 841 CD3+CD4+ and CD26-/CD7- lymphocytes, we show that tumor cells in Sézary syndrome and mycosis fungoides (MF) exhibit different phenotypes and trajectories of differentiation. When compared to MF, Sézary cells exhibit narrower repertoires of TCRs and exhibit clonal enrichment. Surprisingly, we identified ≥200 mutations in hematopoietic stem cells from multiple patients with Sézary syndrome. Mutations in key oncogenes were also present in peripheral Sézary cells, which also showed the hallmarks of recent thymic egression. Together our data suggest that CTCL arises from mutated lymphocyte progenitors that acquire TCRs in the thymus, which complete their malignant transformation in the periphery.

A Novel Humanized Immune Stroma PDX Cancer Model for Therapeutic Studies.

Standard preclinical human tumor models lack a human tumor stroma. However, as stroma contributes to therapeutic resistance, the lack of human stroma may make current models less stringent for testing new therapies. To address this, using patient-derived tumor cells, patient derived cancer-associated mesenchymal stem/progenitor cells, and human endothelial cells, we created a Human Stroma-Patient Derived Xenograft (HS-PDX) tumor model. HS-PDX, compared to the standard PDX model, demonstrate greater resistance to targeted therapy and chemotherapy, and better reflect patient response to therapy. Furthermore, HS-PDX can be grown in mice with humanized bone marrow to create humanized immune stroma patient-derived xenograft (HIS-PDX) models. The HIS-PDX model contains human connective tissues, vascular and immune cell infiltrates. RNA sequencing analysis demonstrated a 94-96% correlation with primary human tumor. Using this model, we demonstrate the impact of human tumor stroma on response to CAR-T cell therapy and immune checkpoint inhibitor therapy. We show an immunosuppressive role for human tumor stroma and that this model can be used to identify immunotherapeutic combinations to overcome stromally mediated immunosuppression. Combined, our data confirm a critical role for human stoma in therapeutic response and indicate that HIS-PDX can be an important tool for preclinical drug testing. Statement of Significance: We developed a tumor model with human stromal, vascular, and immune cells. This model mirrors patient response to chemotherapy, targeted therapy, and immunotherapy, and can be used to study therapy resistance.

Actionable spontaneous antibody responses antagonize malignant progression in ovarian carcinoma.

OBJECTIVE: To demonstrate that shared antibody responses in endometriosis and endometriosis-associated ovarian cancer spontaneously antagonize malignant progression and can be leveraged to develop future immunotherapies. METHODS: B cells from cyopreserved clear cell ovarian carcinoma (CCC, n = 2), endometrioid ovarian carcinoma (EC, n = 2), and endometriomas (n = 2) were isolated, activated, and EBV-immortalized. Antibodies were purified from B cell supernatants and used for screening arrays containing most of the human proteome. Targets were prioritized based on accessibility (transmembrane or secreted proteins), expression in endometriosis and cancer, and concurrent IgA and IgG responses. We focused on antibodies targeting tumor-promoting syndecan binding protein (SDCBP) to demonstrate anti-tumor activity. Immunoblots and qPCR were performed to assess SDCBP expression in ovarian cancer and endometriosis cell lines and tumor samples. Recombinant IgG4 was generated using the variable heavy and light chains of dominant B cell receptors (BCRs) reacting against the extracellular domain of SDCBP, and used in in vivo studies in human CCC- and high-grade serous ovarian carcinoma (HGSOC)-bearing immunodeficient mice. RESULTS: Nine accessible proteins detected by both IgA and IgG were identified in all samples - including SDCBP, which is expressed in ovarian carcinomas of multiple histologies. Administration of α-SDCBP IgG4 in OVCAR3 (HGSOC), TOV21G and RMG-I (CCC) tumor-bearing mice significantly decreased tumor volume compared to control irrelevant IgG4. CONCLUSIONS: Spontaneous antibody responses exert suboptimal but measurable immune pressure against malignant progression in ovarian carcinomas. Using tumor-derived antibodies for developing novel immunotherapeutics warrants further investigation.

Case Report: Durable complete pathologic response and organ preservation following ipilimumab and nivolumab for locally advanced primary vaginal mucosal melanoma.

Optimal management of locally advanced vaginal mucosal melanoma is poorly understood because of its rarity and unique biology. Patients have a poor prognosis despite aggressive management approaches including pelvic exenteration and adjuvant radiation that carry major morbidities. We report a case of a patient in early 40's who experienced complete pathologic response and organ preservation following immunotherapy consisting of 3 cycles of ipilimumab and nivolumab. Treatment was complicated by a high-grade immune mediated hepatitis that eventually resolved with immunosuppressive therapy. Immune monitoring studies utilizing vaginal tumor biopsies showed evidence of enhanced infiltration by CD3+/CD8+ cytotoxic T-cells and increased expression of MHC-I/PD-L1 within the tumor microenvironment following immunotherapy. The patient continues to be without evidence of disease recurrence by radiologic and gynecologic examinations with more than 2 years of follow up from the time of immunotherapy initiation. To our knowledge, this is the only case report in the literature of a patient with locally advanced vaginal mucosal melanoma experiencing a durable complete pathologic response and organ preservation following immune checkpoint blockade as the only treatment approach.

Olfactory Receptor OR2H1 Is an Effective Target for CAR T Cells in Human Epithelial Tumors.

Although chimeric antigen receptor (CAR)-expressing T cells have proven success in hematologic malignancies, their effectiveness in solid tumors has been largely unsuccessful thus far. We found that some olfactory receptors are expressed in a variety of solid tumors of different histologic subtypes, with a limited pattern of expression in normal tissues. Quantification of OR2H1 expression by qRT-PCR and Western blot analysis of 17 normal tissues, 82 ovarian cancers of various histologies, eight non-small cell lung cancers (NSCLCs), and 17 breast cancers demonstrated widespread OR2H1 expression in solid epithelial tumors with expression in normal human tissues limited to the testis. CAR T cells recognizing the extracellular domain of the olfactory receptor OR2H1 were generated with a targeting motif identified through the screening of a phage display library and demonstrated OR2H1-specific cytotoxic killing in vitro and in vivo, using tumor cells with spontaneous expression of variable OR2H1 levels. Importantly, recombinant OR2H1 IgG generated with the VH/VL sequences of the CAR construct specifically detected OR2H1 protein signal in 60 human lung cancers, 40 ovarian carcinomas, and 73 cholangiocarcinomas, at positivity rates comparable with mRNA expression and without OR2H1 staining in 58 normal tissues. CRISPR/Cas9-mediated ablation of OR2H1 confirmed targeting specificity of the CAR and the tumor-promoting role of OR2H1 in glucose metabolism. Therefore, T cells redirected against OR2H1-expressing tumor cells represent a promising therapy against a broad range of epithelial cancers, likely with an admissible toxicity profile.

Ovarian cancer immunogenicity is governed by a narrow subset of progenitor tissue-resident memory T cells.

Despite repeated associations between T cell infiltration and outcome, human ovarian cancer remains poorly responsive to immunotherapy. We report that the hallmarks of tumor recognition in ovarian cancer-infiltrating T cells are primarily restricted to tissue-resident memory (TRM) cells. Single-cell RNA/TCR/ATAC sequencing of 83,454 CD3+CD8+CD103+CD69+ TRM cells and immunohistochemistry of 122 high-grade serous ovarian cancers shows that only progenitor (TCF1low) tissue-resident T cells (TRMstem cells), but not recirculating TCF1+ T cells, predict ovarian cancer outcome. TRMstem cells arise from transitional recirculating T cells, which depends on antigen affinity/persistence, resulting in oligoclonal, trogocytic, effector lymphocytes that eventually become exhausted. Therefore, ovarian cancer is indeed an immunogenic disease, but that depends on ∼13% of CD8+ tumor-infiltrating T cells (∼3% of CD8+ clonotypes), which are primed against high-affinity antigens and maintain waves of effector TRM-like cells. Our results define the signature of relevant tumor-reactive T cells in human ovarian cancer, which could be applicable to other tumors with unideal mutational burden.

TGF-ß-mediated silencing of genomic organizer SATB1 promotes Tfh cell differentiation and formation of intra-tumoral tertiary lymphoid structures.

The immune checkpoint receptor PD-1 on T follicular helper (Tfh) cells promotes Tfh:B cell interactions and appropriate positioning within tissues. Here, we examined the impact of regulation of PD-1 expression by the genomic organizer SATB1 on Tfh cell differentiation. Vaccination of CD4CreSatb1f/f mice enriched for antigen-specific Tfh cells, and TGF-ß-mediated repression of SATB1 enhanced Tfh differentiation of human T cells. Mechanistically, high Icos expression in Satb1-/- CD4+ T cells promoted Tfh cell differentiation by preventing T follicular regulatory cell skewing and resulted in increased isotype-switched B cell responses in vivo. Ovarian tumors in CD4CreSatb1f/f mice accumulated tumor antigen-specific, LIGHT+CXCL13+IL-21+ Tfh cells and tertiary lymphoid structures (TLS). TLS formation decreased tumor growth in a CD4+ T cell and CXCL13-dependent manner. The transfer of Tfh cells, but not naive CD4+ T cells, induced TLS at tumor beds and decreased tumor growth. Thus, TGF-ß-mediated silencing of Satb1 licenses Tfh cell differentiation, providing insight into the genesis of TLS within tumors.

IgA-Dominated Humoral Immune Responses Govern Patients' Outcome in Endometrial Cancer.

Recent studies suggest that B cells could play an important role in the tumor microenvironment. However, the role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we evaluated the role of coordinated humoral and cellular adaptive immune responses in endometrial cancer. Concomitant accumulation of T, B, and plasma cells at tumor beds predicted better survival. However, only B-cell markers corresponded with prolonged survival specifically in high-grade endometrioid type and serous tumors. Immune protection was associated with class-switched IgA and, to a lesser extent, IgG. Expressions of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA were superior predictors of outcome and correlated with defects in methyl-directed DNA mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drove activation of inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and endoplasmic reticulum stress pathways, while thwarting DNA repair mechanisms. Together, these findings suggest that coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer as well as the potential for effective immunotherapies. SIGNIFICANCE: This study provides new insights into the crucial role of humoral immunity in human endometrial cancer, providing a rationale for designing novel immunotherapies against this prevalent malignancy. See related commentary by Osorio and Zamarin, p. 766.

Plasma cell dependence on histone/protein deacetylase 11 reveals a therapeutic target in multiple myeloma.

The clinical utility of histone/protein deacetylase (HDAC) inhibitors in combinatorial regimens with proteasome inhibitors for patients with relapsed and refractory multiple myeloma (MM) is often limited by excessive toxicity due to HDAC inhibitor promiscuity with multiple HDACs. Therefore, more selective inhibition minimizing off-target toxicity may increase the clinical effectiveness of HDAC inhibitors. We demonstrated that plasma cell development and survival are dependent upon HDAC11, suggesting this enzyme is a promising therapeutic target in MM. Mice lacking HDAC11 exhibited markedly decreased plasma cell numbers. Accordingly, in vitro plasma cell differentiation was arrested in B cells lacking functional HDAC11. Mechanistically, we showed that HDAC11 is involved in the deacetylation of IRF4 at lysine103. Further, targeting HDAC11 led to IRF4 hyperacetylation, resulting in impaired IRF4 nuclear localization and target promoter binding. Importantly, transient HDAC11 knockdown or treatment with elevenostat, an HDAC11-selective inhibitor, induced cell death in MM cell lines. Elevenostat produced similar anti-MM activity in vivo, improving survival among mice inoculated with 5TGM1 MM cells. Elevenostat demonstrated nanomolar ex vivo activity in 34 MM patient specimens and synergistic activity when combined with bortezomib. Collectively, our data indicated that HDAC11 regulates an essential pathway in plasma cell biology establishing its potential as an emerging theraputic vulnerability in MM.

Exposure to a mycovirus containing Aspergillus Flavus reproduces acute lymphoblastic leukemia cell surface and genetic markers in cells from patients in remission and not controls.

The etiology of acute lymphoblastic leukemia (ALL) remains unknown. A recent "two-hit" model for the occurrence of precursor B cell acute lymphoblastic leukemia propose that this disease arises through a two-step process, including predisposing genetic mutation and exposure to infections. While several genetic mutations are proposed, no infection category has been suggested. We have isolated a certain Aspergillus Flavus from residence of an ALL patient. This organism contains mycovirus and does not produce aflatoxin. The supernatant of culture of this mycovirus containing Aspergillus Flavus (SAF) was tested on the PBMCs of ALL patients in remission and controls. Cell surface phenotypes and genetic markers were examined. The effects of its combination with Epstein-Barr virus (EBV) was also investigated. For the SAF, positive and negative controls were aflatoxin and culture of Mycocladus corymbifer, respectively. Controls for ALL were sickle cell patients undergoing exchange transfusion. Incubation of the PMBCs from ALL patients in remission, or controls, with SAF resulted in re-development of ALL cell surface phenotypes and genetic markers in ALL patients in remission and not controls. These differentiating effects were not seen with aflatoxin or culture of Mycocladus Corymbifer. Addition of EBV did not alter effects of SAF. Currently, there are no techniques to discriminately reproduce characteristic leukemic genetic markers and cell surface phenotypes in cells from ALL patients in remission and not controls. These studies may provide a test for recognition of ALL patients in remission and new prospects for the investigation of leukemogenesis.

HDAC6 Inhibition Alleviates CLL-Induced T-Cell Dysfunction and Enhances Immune Checkpoint Blockade Efficacy in the Eµ-TCL1 Model.

Development of chronic lymphocytic leukemia (CLL) is associated with severe immune dysfunction. T-cell exhaustion, immune checkpoint upregulation, and increase of regulatory T cells contribute to an immunosuppressive tumor microenvironment. As a result, CLL patients are severely susceptible to infectious complications that increase morbidity and mortality. CLL B-cell survival is highly dependent upon interaction with the supportive tumor microenvironment. It has been postulated that the reversal of T-cell dysfunction in CLL may be beneficial to reduce tumor burden. Previous studies have also highlighted roles for histone deacetylase 6 (HDAC6) in regulation of immune cell phenotype and function. Here, we report for the first time that HDAC6 inhibition exerts beneficial immunomodulatory effects on CLL B cells and alleviates CLL-induced immunosuppression of CLL T cells. In the Eµ-TCL1 adoptive transfer murine model, genetic silencing or inhibition of HDAC6 reduced surface expression of programmed death-ligand 1 (PD-L1) on CLL B cells and lowered interleukin-10 (IL-10) levels. This occurred concurrently with a bolstered T-cell phenotype, demonstrated by alteration of coinhibitory molecules and activation status. Analysis of mice with similar tumor burden indicated that the majority of T-cell changes elicited by silencing or inhibition of HDAC6 in vivo are likely secondary to decrease of tumor burden and immunomodulation of CLL B cells. The data reported here suggest that CLL B cell phenotype may be altered by HDAC6-mediated hyperacetylation of the chaperone heat shock protein 90 (HSP90) and subsequent inhibition of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. Based on the beneficial immunomodulatory activity of HDAC6 inhibition, we rationalized that HDAC6 inhibitors could enhance immune checkpoint blockade in CLL. Conclusively, combination treatment with ACY738 augmented the antitumor efficacy of anti-PD-1 and anti-PD-L1 monoclonal antibodies in the Eµ-TCL1 adoptive transfer murine model. These combinatorial antitumor effects coincided with an increased cytotoxic CD8+ T-cell phenotype. Taken together, these data highlight a role for HDAC inhibitors in combination with immunotherapy and provides the rationale to investigate HDAC6 inhibition together with immune checkpoint blockade for treatment of CLL patients.

The dual PI3Kδ/CK1ε inhibitor umbralisib exhibits unique immunomodulatory effects on CLL T cells.

The in-clinic phosphatidylinositol 3-kinase (PI3K) inhibitors idelalisib (CAL-101) and duvelisib (IPI-145) have demonstrated high rates of response and progression-free survival in clinical trials of B-cell malignancies, such as chronic lymphocytic leukemia (CLL). However, a high incidence of adverse events has led to frequent discontinuations, limiting the clinical development of these inhibitors. By contrast, the dual PI3Kδ/casein kinase-1-ε (CK1ε) inhibitor umbralisib (TGR-1202) also shows high rates of response in clinical trials but has an improved safety profile with fewer severe adverse events. Toxicities typical of this class of PI3K inhibitors are largely thought to be immune mediated, but they are poorly characterized. Here, we report the effects of idelalisib, duvelisib, and umbralisib on regulatory T cells (Tregs) on normal human T cells, T cells from CLL patients, and T cells in an Eµ-TCL1 adoptive transfer mouse CLL model. Ex vivo studies revealed differential effects of these PI3K inhibitors; only umbralisib treatment sustained normal and CLL-associated FoxP3+ human Tregs. Further, although all 3 inhibitors exhibit antitumor efficacy in the Eµ-TCL1 CLL model, idelalisib- or duvelisib-treated mice displayed increased immune-mediated toxicities, impaired function, and reduced numbers of Tregs, whereas Treg number and function were preserved in umbralisib-treated CLL-bearing mice. Finally, our studies demonstrate that inhibition of CK1ε can improve CLL Treg number and function. Interestingly, CK1ε inhibition mitigated impairment of CLL Tregs by PI3K inhibitors in combination treatment. These results suggest that the improved safety profile of umbralisib is due to its role as a dual PI3Kδ/CK1ε inhibitor that preserves Treg number and function.

Plasma of Acute Lymphoblastic Leukemia Patients React to the Culture of a Mycovirus Containing Aspergillus flavus.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and is also seen in adults. Currently, no plasma-based test for the detection of ALL is available. We have cultured the home of a patient with ALL and isolated a mycovirus containing Aspergillus flavus. This culture was subjected to electron microscopy, purification, and mass spectrometry. Using enzyme-linked immunosorbent assay technique, plasma of patients with ALL and long-term survivors of this disease were tested for antibodies, utilizing supernatant of the culture of this organism. The results were compared with 3 groups of controls, including healthy individuals, patients with sickle cell disease, and solid tumors. Using electron microscopy, the isolated A. flavus contained mycovirus particles. In chemical analysis, this organism did not produce any aflatoxin. Using an enzyme-linked immunosorbent assay technique, the supernatant of the culture of the mycovirus containing A. flavus could differentiate ALL patients from each group of controls (P<0.001). These studies provide a new technique for the detection of ALL and may add information for future research regarding leukemogenesis.

HDAC11 deficiency disrupts oncogene-induced hematopoiesis in myeloproliferative neoplasms.

Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.

Silencing of HDAC6 as a therapeutic target in chronic lymphocytic leukemia.

Although the treatment paradigm for chronic lymphocytic leukemia (CLL) is rapidly changing, the disease remains incurable, except with allogeneic bone marrow transplantation, and resistance, relapsed disease, and partial responses persist as significant challenges. Recent studies have uncovered roles for epigenetic modification in the regulation of mechanisms contributing to malignant progression of CLL B cells. However, the extent to which epigenetic modifiers can be targeted for therapeutic benefit in CLL patients remains poorly explored. We report for the first time that expression of epigenetic modifier histone deacetylase 6 (HDAC6) is upregulated in CLL patient samples, cell lines, and euTCL1 transgenic mouse models compared with HDAC6 in normal controls. Genetic silencing of HDAC6 conferred survival benefit in euTCL1 mice. Administration of isoform-specific HDAC6 inhibitor ACY738 in the euTCL1 aging and adoptive transfer models deterred proliferation of CLL B cells, delayed disease onset via disruption of B-cell receptor signaling, and sensitized CLL B cells to apoptosis. Furthermore, coadministration of ACY738 and ibrutinib displayed synergistic cell kill against CLL cell lines and improved overall survival compared with either single agent in vivo. These results demonstrate for the first time the therapeutic efficacy of selective HDAC6 inhibition in preclinical CLL models and suggest a rationale for the clinical development of HDAC6 inhibitors for CLL treatment, either alone or in combination with Bruton tyrosine kinase inhibition.

Essential role for histone deacetylase 11 (HDAC11) in neutrophil biology.

Epigenetic changes in chromatin structure have been recently associated with the deregulated expression of critical genes in normal and malignant processes. HDAC11, the newest member of the HDAC family of enzymes, functions as a negative regulator of IL-10 expression in APCs, as previously described by our lab. However, at the present time, its role in other hematopoietic cells, specifically in neutrophils, has not been fully explored. In this report, for the first time, we present a novel physiologic role for HDAC11 as a multifaceted regulator of neutrophils. Thus far, we have been able to demonstrate a lineage-restricted overexpression of HDAC11 in neutrophils and committed neutrophil precursors (promyelocytes). Additionally, we show that HDAC11 appears to associate with the transcription machinery, possibly regulating the expression of inflammatory and migratory genes in neutrophils. Given the prevalence of neutrophils in the peripheral circulation and their central role in the first line of defense, our results highlight a unique and novel role for HDAC11. With the consideration of the emergence of new, selective HDAC11 inhibitors, we believe that our findings will have significant implications in a wide range of diseases spanning malignancies, autoimmunity, and inflammation.

Histone deacetylase 11: A novel epigenetic regulator of myeloid derived suppressor cell expansion and function.

Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells capable of suppressing anti-tumor T cell function in the tumor microenvironment, represent an imposing obstacle in the development of cancer immunotherapeutics. Thus, identifying elements essential to the development and perpetuation of these cells will undoubtedly improve our ability to circumvent their suppressive impact. HDAC11 has emerged as a key regulator of IL-10 gene expression in myeloid cells, suggesting that this may represent an important targetable axis through which to dampen MDSC formation. Using a murine transgenic reporter model system where eGFP expression is controlled by the HDAC11 promoter (Tg-HDAC11-eGFP), we provide evidence that HDAC11 appears to function as a negative regulator of MDSC expansion/function in vivo. MDSCs isolated from EL4 tumor-bearing Tg-HDAC11-eGFP display high expression of eGFP, indicative of HDAC11 transcriptional activation at steady state. In striking contrast, immature myeloid cells in tumor-bearing mice display a diminished eGFP expression, implying that the transition of IMC to MDSC's require a decrease in the expression of HDAC11, where we postulate that it acts as a gate-keeper of myeloid differentiation. Indeed, tumor-bearing HDAC11-knockout mice (HDAC11-KO) demonstrate a more suppressive MDSC population as compared to wild-type (WT) tumor-bearing control. Notably, the HDAC11-KO tumor-bearing mice exhibit enhanced tumor growth kinetics when compare to the WT control mice. Thus, through a better understanding of this previously unknown role of HDAC11 in MDSC expansion and function, rational development of targeted epigenetic modifiers may allow us to thwart a powerful barrier to efficacious immunotherapies.

Overexpression of TCL1 activates the endoplasmic reticulum stress response: a novel mechanism of leukemic progression in mice.

Chronic lymphocytic leukemia (CLL) represents 30% of adult leukemia. TCL1 is expressed in ~ 90% of human CLL. Transgenic expression of TCL1 in murine B cells (Eµ-TCL1) results in mouse CLL. Here we show for the first time that the previously unexplored endoplasmic reticulum (ER) stress response is aberrantly activated in Eµ-TCL1 mouse and human CLL. This includes activation of the IRE-1/XBP-1 pathway and the transcriptionally up-regulated expression of Derlin-1, Derlin-2, BiP, GRP94, and PDI. TCL1 associates with the XBP-1 transcription factor, and causes the dysregulated expression of the transcription factors, Pax5, IRF4, and Blimp-1, and of the activation-induced cytidine deaminase. In addition, TCL1-overexpressing CLL cells manufacture a distinctly different BCR, as we detected increased expression of membrane-bound IgM and altered N-linked glycosylation of Igα and Igß, which account for the hyperactive BCR in malignant CLL. To demonstrate that the ER stress-response pathway is a novel molecular target for the treatment of CLL, we blocked the IRE-1/XBP-1 pathway using a novel inhibitor, and observed apoptosis and significantly stalled growth of CLL cells in vitro and in mice. These studies reveal an important role of TCL1 in activating the ER stress response in support for malignant progression of CLL.

Epigenetic repolarization of T lymphocytes from chronic lymphocytic leukemia patients using 5-aza-2'-deoxycytidine.

T cell immune dysfunction has an important role in the profound immune suppression that characterizes chronic lymphocytic leukemia (CLL). Improper polarization of T cells has been proposed as one of the mechanism involved. Mounting data implicates chromatin regulation, namely promoter methylation, in the plasticity of naïve human T cells. Recent in vitro evidence indicates that this plasticity may be phenotypically altered by using methylation inhibitors which are approved for clinical use in certain types of cancer. These results beg the question: can the ineffective polarization of T lymphocytes in the context of CLL be effectively modulated using methylation inhibitors in a sustainable therapeutic fashion? To answer this question our laboratory has studied the effects of 5-aza-2'-deoxycytidine (5A2) in helper and cytotoxic T lymphocytes from healthy donors and CLL patients in well characterized molecular and epigenetic signaling pathways involved in effective polarization. Moreover, we sought to investigate the consequences of methylation inhibitor treatment on lymphocyte survival, activation intensity, and naïve cell polarization. Our data indicates that 5A2 treatment can depolarize Th2 cells to effectively secrete interferon gamma, signal via T-bet, and achieve demethylation of critical Th1 specific promoters. Moreover, we demonstrate that 5A2 can force Th1 polarization of naïve T cells despite a strong IL-4 stimuli and a lack of IL-12. In conclusion our data seeks to define a modality in which improper or ineffective T cell polarization can be altered by 5AZA and could be incorporated in future therapeutic interventions.