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Purpose: Missense mutated von Hippel Lindau (VHL) protein (pVHL) maintains intrinsic function but undergoes proteasomal degradation and tumor initiation and/or progression in VHL disease. Vorinostat can rescue missense mutated pVHL and arrest tumor growth in preclinical models. We asked whether short-term oral vorinostat could rescue pVHL in central nervous system hemangioblastomas in patients with germline missense VHL. Patients and Methods: We administered oral vorinostat to 7 subjects (ages 46.0 ± 14.5 years) and then removed symptomatic hemangioblastomas surgically (ClinicalTrials.gov identifier NCT02108002). Results: Vorinostat was tolerated without serious adverse events by all patients. pVHL expression was elevated in neoplastic stromal cells compared with untreated hemangioblastomas from same patients. We found transcriptional suppression of downstream hypoxia-inducible factor (HIF) effectors. Mechanistically, vorinostat prevented Hsp90 recruitment to mutated pVHL in vitro. The effects of vorinostat on the Hsp90–pVHL interaction, pVHL rescue, and transcriptional repression of downstream HIF effectors was independent of the location of the missense mutation on the VHL locus. We confirmed a neoplastic stromal cell–specific effect in suppression of protumorigenic pathways with single-nucleus transcriptomic profiling. Conclusions: We found that oral vorinostat treatment in patients with germline missense VHL mutations has a potent biologic effect that warrants further clinical study. These results provide biologic evidence to support the use of proteostasis modulation for the treatment of syndromic solid tumors involving protein misfolding. Proteostasis modulation with vorinostat rescues missense mutated VHL protein. Further clinical trials are needed to demonstrate tumor growth arrest.

Protein phosphatases play essential roles as negative regulators of kinases and signaling cascades involved in cytoskeletal organization. Protein phosphatase 2A (PP2A) is highly conserved and is the predominant serine/threonine phosphatase in the nervous system, constituting more than 70% of all neuronal phosphatases. PP2A is involved in diverse regulatory functions, including cell cycle progression, apoptosis, and DNA repair. Although PP2A has historically been identified as a tumor suppressor, inhibition of PP2A has paradoxically demonstrated potential as a therapeutic target for various cancers. LB100, a water-soluble, small-molecule competitive inhibitor of PP2A, has shown particular promise as a chemo- and radio-sensitizing agent. Preclinical success has led to a profusion of clinical trials on LB100 adjuvant therapies, including a phase I trial in extensive-stage small-cell lung cancer, a phase I/II trial in myelodysplastic syndrome, a phase II trial in recurrent glioblastoma, and a completed phase I trial assessing the safety of LB100 and docetaxel in various relapsed solid tumors. Herein, we review the development of LB100, the role of PP2A in cancer biology, and recent advances in targeting PP2A inhibition in immunotherapy.

Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths, and its therapy remains a challenge. Our proposed therapeutic approach is based on the intratumoral injections of mannan-BAM, toll-like receptor ligands, and anti-CD40 antibody (thus termed MBTA therapy), and has shown promising results in the elimination of subcutaneous murine melanoma, pheochromocytoma, colon carcinoma, and smaller pancreatic adenocarcinoma (Panc02). Here, we tested the short- and long-term effects of MBTA therapy in established subcutaneous Panc02 tumors two times larger than in previous study and bilateral Panc02 models as well as the roles of CD4(+) and CD8(+) T lymphocytes in this therapy. The MBTA therapy resulted in eradication of 67% of Panc02 tumors with the development of long-term memory as evidenced by the rejection of Panc02 cells after subcutaneous and intracranial transplantations. The initial Panc02 tumor elimination is not dependent on the presence of CD4(+) T lymphocytes, although these cells seem to be important in long-term survival and resistance against tumor retransplantation. The resistance was revealed to be antigen-specific due to its inability to reject B16-F10 melanoma cells. In the bilateral Panc02 model, MBTA therapy manifested a lower therapeutic response. Despite numerous combinations of MBTA therapy with other therapeutic approaches, our results show that only simultaneous application of MBTA therapy into both tumors has potential for the treatment of the bilateral Panc02 model.

Cisplatin is currently used to treat inoperable recurrent meningiomas, but its side effects and drug resistance limit its use. Metformin has recently been identified as a chemosensitizing agent. However, the combined treatment of cisplatin and metformin in high-grade meningiomas has not been reported. Herein, our findings demonstrate metformin significantly enhanced cisplatin-induced inhibition of proliferation in meningioma cells, which was associated with the induction of G0/G1 cell cycle arrest. Additionally, metformin activated adenosine monophosphate activated protein kinase (AMPK) and repressed the mammalian target of rapamycin (mTOR) signaling pathways via an AMPK-dependent mechanism. Furthermore, our xenograft murine model confirmed that metformin enhanced cisplatin’s anti-cancer effect by upregulation of AMPK and downregulation of mTOR signaling pathways. In addition, in 63 patients with atypical meningiomas, the activation of AMPK was significantly associated with tumor recurrence and short disease-free survival (DFS). These results demonstrate metformin enhanced the anti-cancer effect of cisplatin in meningioma in vitro and in vivo, an effect mediated through the activation of AMPK and repression of mTOR signaling pathways. Our study suggests the combined treatment of metformin and cisplatin is an effective and safe treatment for high-grade meningiomas., Graphical Abstract, Lin, Zhuang, and their scholars confirmed for the first time that metformin combined with cisplatin is a safe and effective treatment for high-grade meningioma. This study illuminates a new direction for treating high-grade meningioma.

Originally approved in 1979, a specific grading classification for central nervous system (CNS) tumors was devised by the World Health Organization (WHO) in an effort to guide cancer treatment and better understand prognosis. These “blue books” have since undergone several iterations based on tumor location, advancements in histopathology, and most recently, diagnostic molecular pathology in its fifth edition. As new research methods have evolved to elucidate complex molecular mechanisms of tumorigenesis, a need to update and integrate these findings into the WHO grading scheme has become apparent. Epigenetic tools represent an area of burgeoning interest that encompasses all non-Mendelian inherited genetic features affecting gene expression, including but not limited to chromatin remodeling complexes, DNA methylation, and histone regulating enzymes. The SWItch/Sucrose non-fermenting (SWI/SNF) chromatin remodeling complex is the largest mammalian family of chromatin remodeling proteins and is estimated to be altered in 20–25% of all human malignancies; however, the ways in which it contributes to tumorigenesis are not fully understood. We recently discovered that CNS tumors with SWI/SNF mutations have revealed an oncogenic role for endogenous retroviruses (ERVs), remnants of exogenous retroviruses that integrated into the germline and are inherited like Mendelian genes, several of which retain open reading frames for proteins whose expression putatively contributes to tumor formation. Herein, we analyzed the latest WHO classification scheme for all CNS tumors with documented SWI/SNF mutations and/or aberrant ERV expression, and we summarize this information to highlight potential research opportunities that could be integrated into the grading scheme to better delineate diagnostic criteria and therapeutic targets.

Despite the advances made in cancer treatment over the past decades, one statistic has remained unchanged: metastatic cancer accounts for 90 percent of annual cancer deaths in the United States. Our group previously demonstrated that an autologous whole tumor cell vaccine (rWTC-MBTA: irradiated autologous whole tumor cells pulsed with Mannan-BAM, TLR ligands, and anti-CD40 antibody) had a potent anti-tumor immune response and prolonged survival in a mouse colon carcinoma model. To investigate whether rWTC-MBTA would work in “cold” tumor models, we evaluated the vaccine’s effect on preventing and treating tumor metastasis in 4T1 breast tumors. Our data showed that vaccinated mice could significantly prevent lung metastasis in both intravenous injection and mammary pad subcutaneous implantation animal models. Further, we used another metastasis model that more closely mimics clinical practice by resecting the primary tumor after metastasis development. The data showed that vaccinated mice could prevent tumor recurrence, increase T-cell infiltration in the metastatic tumor, and prolong the survival curve. The mechanistic investigation by immunophenotyping revealed that rWTC-MBTA vaccination induced both effector memory (CD44+CD62L−) and center memory (CD44+CD62L+) T cells as well as increased overall CD4+ and CD8+ T-cell count while depletion experiments demonstrated that CD8+ T cells were required for vaccine efficacy. Isolated splenocytes co-cultured with 4T1 cells showed rWTC-MBTA significantly increased T-cell mediated cytotoxicity through TNF-a and IFN-γ in CD107a+CD4+ T cells and Granzyme B and IFN-γ in CD107a+CD8+ T cells. In all experiments, vaccination exerted negligible systemic toxicity. Collectively, our study demonstrates that the rWTC-MBTA vaccine is a safe and promising therapeutic option to prevent and treat tumor metastasis by triggering an antitumor immune response. Citation Format: Juan Ye, Herui Wang, Mitchell Sun, Ondrej Uher, Mark R. Gilbert, Karel Pacak, Zhengping Zhuang, Rogelio Medina, Samik Chakraborty, Jan Zenka. Unique autologous cancer vaccine comprised of irradiated whole tumor cells and MBTA (rWTC-MBTA) triggers antitumor immune response to prevent metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6800.

Despite numerous therapeutic advances in cancers, the treatment of glioblastoma multiforme (GBM) remains a challenge. Boosting T-lymphocyte mediated responses against GBM offers a promising approach towards solving this problem. Herein, we present a therapeutic vaccination strategy that promotes the phagocytosis of tumor cells, enhances tumor antigen presentation, and induces a tumor-specific adaptive immune response with subsequent tumor eradication. This strategy consists of subcutaneous injection of irradiated whole tumor cells (rWTC) pulsed with phagocytic agonists (Mannan-BAM), TLR ligands (LTA, Poly (I:C), and R-848), and anti-CD40 agonistic antibody (collectively abbreviated as rWTC-MBTA). We evaluated the therapeutic efficacy of rWTC-MBTA strategy in mouse syngeneic GBM tumor models with GL261 and SB28 cells. In GL261 GBM model, complete regression (CR) of intracranial tumors was achieved in 70% (7/10) of rWTC-MBTA treated animals while none survived in the control group. Of note, the therapeutic efficacy of rWTC-MBTA was abolished in CD4-T and/or CD8-T lymphocyte depleted mice. Immunophenotyping analyses of peripheral lymph nodes and brain tumors of rWTC-MBTA treated mice demonstrated increased antigen presenting cells (dendritic cells and MHC II+ monocytes) and increased cytotoxic IFNγ, TNFα, and granzyme B-secreting CD4-T and CD8-T cells. All three CR mice that were rechallenged with GL261 cells intracranially 14 months after their last rWTC-MBTA treatment resisted tumor development, confirming the establishment of long-term immunological memory. In SB28 GBM model, 80% (8/10) rWTC-MBTA treated mice survived past 95 days after tumor cell implantation without any GBM-related symptoms, with median survival being only 35 days in control groups. In summary, our study demonstrated that rWTC-MBTA strategy can induce potent adaptive immune response against GBM in pre-clinical models. Citation Format: Herui Wang, Rogelio Medina, Juan Ye, Samik Chakraborty, Ondrej Uher, Mitchell Sun, Jan Zenka, Mark R. Gilbert, Karel Pacak, Zhengping Zhuang. Irradiated whole tumor cells pulsed with mannan-BAM, TLR ligands and anti-CD40 antibody serve as a potent tumor cell vaccine against glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 691.

Hypoxia-inducible factors (HIFs) regulate oxygen sensing and expression of genes involved in angiogenesis and erythropoiesis. Polycythemia has been observed in patients with hepatocellular carcinoma (HCC), but the underlying molecular basis remains unknown. Liver tissues from 302 HCC patients, including 104 with polycythemia, were sequenced for HIF2A mutations. A germline HIF2A mutation was detected in one HCC patient with concurrent polycythemia. Three additional family members carried this mutation, but none exhibited polycythemia or were diagnosed with HCC. The gain-of-function mutation resulted in a HIF-2α protein that was transcribed normally but resistant to degradation. HIF-2α target genes EDN1, EPO, GNA14, and VEGF were significantly upregulated in the tumor bed but not in the surrounding liver tissue. Polycythemia resolved upon total resection of the tumor tissue. This newly described HIF2A mutation may promote HCC oncogenesis.

Zinc fingers and homeoboxes 1 (ZHX1) is a transcription repressor that has been implicated in the tumorigenesis and progression of diverse tumors. The functional role and regulating mechanism of ZHX1 has not been elucidated in glioblastoma (GBM). Previous reports have suggested that a large number of non-coding RNAs play a vital role in glioma initiation and progression. This study aimed to investigate the functional role and co-regulatory mechanisms of the metastasis-associated lung adenocarcinoma transcript-1 (MALAT1)/ microRNA-199a (miR-199a)/ZHX1 axis in GBM. We analyzed the expression of the MALAT1/miR-199a/ZHX1 axis and its correlation with patients’ overall survival using two different glioma gene-expression datasets. A series of in vitro and in vivo studies including dual luciferase reporter assay, fluorescence in situ hybridization (FISH), RNA immunoprecipitation, and pull-down experiments were completed to elucidate the biological significance of the MALAT1/miR-199a/ZHX1 axis in promoting glioma proliferation and progression. Elevated ZHX1 expression correlated with poor prognosis in GBM patients, and in vitro studies demonstrated that ZHX1 attenuated GBM cell apoptosis by downregulation of pro-apoptotic protein (Bax) and upregulation of anti-apoptotic protein (Bcl-2). Furthermore, knockdown of MALAT1 inhibited GBM proliferation and progression in vitro and reduced tumor volume and prolonged survival in an orthotopic GBM murine model. Finally, we demonstrated that MALAT1 promoted ZHX1 expression via acting as a competing endogenous RNA by sponging miR-199a. The MALAT1/miR-199a/ZHX1 axis promotes GBM cell proliferation and progression in vitro and in vivo, and its expression negatively correlates with GBM patient survival. Blocking the MALAT1/miR-199a/ZHX1 axis can serve as a novel therapeutic strategy for treating GBM. Keywords: MALAT1, ZHX1, glioblastoma, competing endogenous RNA, long non-coding RNA

Background Compared to normal cells, cancer cells exhibit a higher level of oxidative stress, which primes key cellular and metabolic pathways and thereby increases their resilience under oxidative stress. This higher level of oxidative stress also can be exploited to kill tumor cells while leaving normal cells intact. In this study we have found that isovalerylspiramycin I (ISP I), a novel macrolide antibiotic, suppresses cancer cell growth and tumor metastases by targeting the nucleolar protein selenoprotein H (SELH), which plays critical roles in keeping redox homeostasis and genome stability in cancer cells. Methods We developed ISP I through genetic recombination and tested the antitumor effects using primary and metastatic cancer models. The drug target was identified using the drug affinity responsive target stability (DARTS) and mass spectrum assays. The effects of ISP I were assessed for reactive oxygen species (ROS) generation, DNA damage, R-loop formation and its impact on the JNK2/TIF-IA/RNA polymerase I (POLI) transcription pathway. Results ISP I suppresses cancer cell growth and tumor metastases by targeting SELH. Suppression of SELH induces accumulation of ROS and cancer cell-specific genomic instability. The accumulation of ROS in the nucleolus triggers nucleolar stress and blocks ribosomal RNA transcription via the JNK2/TIF-IA/POLI pathway, causing cell cycle arrest and apoptosis in cancer cells. Conclusions We demonstrated that ISP I links cancer cell vulnerability to oxidative stress and RNA biogenesis by targeting SELH. This suggests a potential new cancer treatment paradigm, in which the primary therapeutic agent has minimal side-effects and hence may be useful for long-term cancer chemoprevention.

Despite numerous therapeutic advances, the treatment of glioblastoma multiforme (GBM) remains a challenge, with current 5-year survival rates estimated at 4%. Multiple characteristic elements of GBM contribute to its treatment-resistance, including its low immunogenicity and its highly immunosuppressive microenvironment that can effectively disarm adaptive immune responses. Hence, therapeutic strategies that aim to boost T-lymphocyte mediated responses against GBM are of great therapeutic value. Herein, we present a therapeutic vaccination strategy that promotes the phagocytosis of tumor cells, enhances tumor antigen presentation, and induces a tumor-specific adaptive immune response. This strategy consists of vaccinations with irradiated whole tumor cells (rWTC) pulsed with phagocytic agonists (Mannan-BAM), TLR ligands [LTA, Poly (I:C), and R-848], and anti-CD40 antibody (collectively abbreviated as rWTC-MBTA). We evaluated the therapeutic efficacy of rWTC-MBTA strategy in a mouse syngeneic GL261 orthotopic GBM tumor model. rWTC-MBTA or vehicle control were administered subcutaneously over the right foreleg three days after intracranial injection of GL261 cells. Complete regression (CR) of intracranial tumors was achieved in 70% (7/10) of rWTC-MBTA treated animals while none survived in the control group. Immunophenotyping analyses of peripheral lymph nodes and brain tumors of rWTC-MBTA treated mice demonstrated: (1) increased mature dendritic cells and MHC II+ monocytes; (2) increased effector (CD62L-CD44+) CD4-T and CD8-T cells; (3) increased cytotoxic IFNγ-, TNFα-, and granzyme B-secreting CD4-T and CD8-T cells. Of note, the therapeutic efficacy of rWTC-MBTA disappeared in CD4-T and/or CD8-T lymphocyte depleted mice. Three mice that achieved CR were rechallenged with 50k GL261 cells intracranially 14 months after the last rWTC-MBTA treatment and all rechallenged animals resisted GL261 tumor development, confirming the establishment of long-term immunological memory against GL261 tumor cells. Collectively, our study demonstrated that rWTC-MBTA strategy can effectively activate antigen presenting cells and induce more favorable T-cell signatures in the GBM tumors.

Glioblastoma (GBM) is the most common and the deadliest type of brain tumor affecting the adult central nervous system, carrying a median survival of 14 months. In vivo syngeneic animal models of GBM that recapitulate human GBM are urgently needed to decipher glioma biology and develop novel therapeutics, including immunotherapy. Here, we generated a syngeneic mouse glioblastoma stem cell line (RCAS-TVA-GSC01) through repeated in-vitro passages of stem-like tumor cells isolated from the RCAS-TVA glioma mouse model, which overexpresses PDGFA and carries a homozygous deletion of Ink4a-Arf locus. We characterized this new GSC line both in vitro and in vivo. This cell line expresses stemness markers CD133 and Nestin, and is able to differentiate into astrocyte, neuron, and oligodendrocyte-like cells in 2% FBS differentiating medium with markers GFAP, β-III tubulin, and O4, respectively. Transcriptomic and genomic profiling of RCAS-TVA-GSC01 cells revealed molecular heterogeneity comparable to human glioblastoma. When intracranially transplanted into wild-type C57BL/6 mice, this cell line formed high-grade gliomas with a median survival time of 56.5 days. The differentiation potential and molecular heterogeneity make RCAS-TVA GSC01 line a unique and useful tool for studying multiple aspects of glioblastoma pathogenesis and a promising platform for testing new GBM therapeutics study, especially for immunotherapy that requires a syngeneic mouse model.

Simple Summary Paclitaxel-based chemotherapy has been introduced to treat esophageal squamous cell carcinoma. While its initial efficacy has been clinically established, the development of resistance is inevitable. To understand the paclitaxel resistance mechanism, we developed nanoparticle albumin-bound paclitaxel (nab-PTX)-resistant esophageal squamous cell lines from their sensitive counterparts. We found that resistant cells express higher levels of protein phosphatase 2A (PP2A), oxidative phosphorylation, and anti-apoptotic protein MCL1 than their counterparts. Therapeutically, the PP2A inhibitor LB-100 synergistically sensitized resistant esophageal squamous cells to nab-PTX both in vitro and in vivo. Therefore, our data suggest that LB-100 may potentially overcome nab-PTX resistance in a clinical setting. Abstract Paclitaxel-based chemotherapy is a treatment option for advanced esophageal squamous cell carcinoma (ESCC). However, the development of chemoresistance leads to treatment failure, and the underlying mechanism remains elusive. We investigated the mechanisms of nanoparticle albumin-bound paclitaxel (nab-PTX) resistance by establishing three nab-PTX resistant ESCC cell lines. Proteomics analysis revealed higher oxidative phosphorylation (OXPHOS) in resistant cell line DR150 than in its parental cell line KYSE150, which is likely caused by stabilized anti-apoptotic protein MCL1. Additionally, we discovered the elevated activity of protein phosphatase 2A (PP2A), the phosphatase that dephosphorylates and stabilizes MCL1, in nab-PTX resistant cell lines. Pharmacological inhibition of PP2A with small molecule compound LB-100 decreased MCL1 protein level, caused more apoptosis in nab-PTX resistant ESCC cell lines than in the parental cells in vitro, and significantly inhibited the tumor growth of nab-PTX resistant xenografts in vivo. Moreover, LB-100 pretreatment partially restored nab-PTX sensitivity in the resistant cell lines and synergistically inhibited the tumor growth of nab-PTX resistant xenografts with nab-PTX. In summary, our study identifies a novel mechanism whereby elevated PP2A activity stabilizes MCL1 protein, increases OXPHOS, and confers nab-PTX resistance, suggesting that targeting PP2A is a potential strategy for reversing nab-PTX resistance in patients with advanced ESCC.

Simple Summary Multiple types of primary tumors and metastases that present with very little if any immune cell infiltration (so-called immunologically “cold” tumors) do not respond to current immunotherapies. In this study, we show that recently developed intratumoral application-based immunotherapy using mannan-BAM, TLR ligands, and anti-CD40 antibody (MBTA therapy) efficiently suppresses tumor growth in a murine bilateral pheochromocytoma model. Moreover, MBTA therapy increases the recruitment of innate immune cells followed by adaptive immune cells not only to primary (injected) tumors but also distal (non-injected) tumors. We also demonstrated that after successful MBTA therapy of subcutaneous pheochromocytoma, long-term immunological memory is driven by CD4+ T cells. Taken together, this study helps to better understand the systemic effect of MBTA therapy and its use for tumor and metastasis reduction or even elimination. Abstract Immunotherapy has become an essential component in cancer treatment. However, the majority of solid metastatic cancers, such as pheochromocytoma, are resistant to this approach. Therefore, understanding immune cell composition in primary and distant metastatic tumors is important for therapeutic intervention and diagnostics. Combined mannan-BAM, TLR ligand, and anti-CD40 antibody-based intratumoral immunotherapy (MBTA therapy) previously resulted in the complete eradication of murine subcutaneous pheochromocytoma and demonstrated a systemic antitumor immune response in a metastatic model. Here, we further evaluated this systemic effect using a bilateral pheochromocytoma model, performing MBTA therapy through injection into the primary tumor and using distant (non-injected) tumors to monitor size changes and detailed immune cell infiltration. MBTA therapy suppressed the growth of not only injected but also distal tumors and prolonged MBTA-treated mice survival. Our flow cytometry analysis showed that MBTA therapy led to increased recruitment of innate and adaptive immune cells in both tumors and the spleen. Moreover, adoptive CD4+ T cell transfer from successfully MBTA-treated mice (i.e., subcutaneous pheochromocytoma) demonstrates the importance of these cells in long-term immunological memory. In summary, this study unravels further details on the systemic effect of MBTA therapy and its use for tumor and metastasis reduction or even elimination.

Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.

Background Pituitary adenomas are common brain tumors. Although transsphenoidal surgery are able to achieve extensive tumor removal, the rate of recurrence ranges from 5 to 20% depending on the different subtype. Further understanding of these tumors is needed to develop novel strategies to improve the prognosis of patients. But their metabolic characteristics are largely unknown. Methods We used metabolomic, transcriptomic, and proteomic approaches to systematically investigate eight subtypes of pituitary adenomas and normal pituitary glands. By blocking IDH2, we investigate IDH2 play an inhibitory role in GH tumor cell growth and tumor secretion. Results We found that all of the pituitary adenomas displayed downregulated glucose metabolism and glycolysis compared to normal tissues. Together with the differences in amino acids and fatty acids, we categorized these tumors into three clusters. We then re-established the reprogrammed metabolic flux in pituitary adenomas based on multiomic analyses. Take growth hormone-secreting pituitary adenomas as an example, we revealed that IDH2 is a key player in the reprogrammed metabolism of such tumors. By blocking IDH2, we confirmed that IDH2 is a potential target for the inhibition of tumor cell growth and tumor secretion. Conclusions Our study first uncovered the metabolic landscape of pituitary adenomas and demonstrated a possible way to inhibit tumor growth by regulating aberrant metabolism.

There is no doubt that immunotherapy lies in the spotlight of current cancer research and clinical trials. However, there are still limitations in the treatment response in certain types of tumors largely due to the presence of the complex network of immunomodulatory and immunosuppressive pathways. These limitations are not likely to be overcome by current immunotherapeutic options, which often target isolated steps in immune pathways preferentially involved in adaptive immunity. Recently, we have developed an innovative anti-cancer immunotherapeutic strategy that initially elicits a strong innate immune response with subsequent activation of adaptive immunity in mouse models. Robust primary innate immune response against tumor cells is induced by toll-like receptor ligands and anti-CD40 agonistic antibodies combined with the phagocytosis-stimulating ligand mannan, anchored to a tumor cell membrane by biocompatible anchor for membrane. This immunotherapeutic approach results in a dramatic therapeutic response in large established murine subcutaneous tumors including melanoma, sarcoma, pancreatic adenocarcinoma, and pheochromocytoma. Additionally, eradication of metastases and/or long-lasting resistance to subsequent re-challenge with tumor cells was also accomplished. Current and future advantages of this immunotherapeutic approach and its possible combinations with other available therapies are discussed in this review.

Background Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. Methods First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. Results We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. Conclusions By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.

Previously we identified the p.Thr61Ile mutation in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) in a Chinese family with autosomal dominant Parkinson’s disease. But the mechanism is still unclear. In this study, we explored the effects of CHCHD2 p.Thr61Ile mutation in cells and its association with coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10). We found that overexpression of Parkinson’s disease–associated T61I mutant CHCHD2 did not produce mitochondrial dysfunction. Rather, its protective effect from stress was abrogated. And, the level of the CHCHD2 protein and mRNA in patient fibroblasts was not significantly different from control. In addition, CHCHD2 T61I mutation caused increased interaction with CHCHD10 and reduced CHCHD10 level. The mitochondrial ultrastructural alterations in CHCHD2 T61I mutant patient fibroblasts are similar to that of CHCHD10 mutations. We therefore propose that CHCHD10 is involved in the development of Parkinson’s disease caused by CHCHD2 loss-of-function mutation p.T61I.

Paragangliomas and pheochromocytomas (PPGLs) are chromaffin tumors associated with severe catecholamine-induced morbidities. Surgical removal is often curative. However, complete resection may not be an option for patients with succinate dehydrogenase subunit A-D (SDHx) mutations. SDHx mutations are associated with a high risk for multiple recurrent, and metastatic PPGLs. Treatment options in these cases are limited and prognosis is dismal once metastases are present. Identification of new therapeutic targets and candidate drugs is thus urgently needed. Previously, we showed elevated expression of succinate receptor 1 (SUCNR1) in SDHB PPGLs and SDHD head and neck paragangliomas. Its ligand succinate has been reported to accumulate due to SDHx mutations. We thus hypothesize that autocrine stimulation of SUCNR1 plays a role in the pathogenesis of SDHx mutation-derived PPGLs. We confirmed elevated SUCNR1 expression in SDHx PPGLs and after SDHB knockout in progenitor cells derived from a human pheochromocytoma (hPheo1). Succinate significantly increased viability of SUCNR1-transfected PC12 and ERK pathway signaling compared to control cells. Candidate SUCNR1 inhibitors successfully reversed proliferative effects of succinate. Our data reveal an unrecognized oncometabolic function of succinate in SDHx PPGLs, providing a growth advantage via SUCNR1.

The foundation of precision immunotherapy in oncology is rooted in computational biology and patient-derived sample sequencing to enrich for and target immunogenic epitopes. Discovery of these tumor-specific epitopes through tumor sequencing has revolutionized patient outcomes in many types of cancers that were previously untreatable. However, these therapeutic successes are far from universal, especially with cancers that carry high intratumoral heterogeneity such as glioblastoma (GBM). Herein, we present the technical aspects of Mannan-BAM, TLR Ligands, Anti-CD40 Antibody (MBTA) vaccine immunotherapy, an investigational therapeutic that potentially circumvents the need for in silico tumor-neoantigen enrichment. We then review the most promising GBM vaccination strategies to contextualize the MBTA vaccine. By reviewing current evidence using translational tumor models supporting MBTA vaccination, we evaluate the underlying principles that validate its clinical applicability. Finally, we showcase the translational potential of MBTA vaccination as a potential immunotherapy in GBM, along with established surgical and immunologic cancer treatment paradigms.

Immunotherapy has become an essential component of cancer treatment, however, a majority of patients with solid metastatic cancers, such as pheochromocytoma (PHEO), do not respond to this type of therapy. Recently, we developed an intratumoral (i.t.) immunotherapy based on the unique combination of TLR ligands, anti-CD40 antibodies, and mannan, which is artificially bound to tumor cells via an anchor (MBTA therapy). This therapy resulted in the complete eradication of aggressive subcutaneous PHEO in 67% of mice and demonstrated a systemic antitumor immune response and regression of non-treated lesions in the metastatic model (1). To further evaluate this systemic effect generated during MBTA therapy, we established a murine bilateral PHEO model, where MBTA therapy was i.t. injected into one tumor, and the distant (non-treated) tumor was monitored for changes in size and immune cell infiltration. The growth of both MBTA-treated and distant tumors was reduced compared to that of the control. Interestingly, survival of the MBTA-treated mice was twice as long compared to the control mice. Moreover, we have made several unique observations during the experiments which were focused on the tumor microenvironment. Flow cytometry analysis revealed the ability of MBTA therapy to significantly increase the infiltration of innate immune cells (monocytes, DCs, macrophages, NK cells) not only in MBTA-treated tumors, but also in distal tumors, despite the fact that MBTA therapy was designed to elicit only local inflammation. An analysis of the macrophage phenotype revealed a switch from protumor M2 to antitumor M1 macrophages in both tumors during the entire MBTA therapy treatment. Analysis of splenic adaptive immune cells revealed that naïve CD4+ or CD8+ T cells differentiated into central memory cells and effector memory cells. CD4+ and CD8+ T cells were elevated in MBTA-treated and distant tumors with a significantly higher frequency of CD8+ effector memory T cells. Moreover, the adoptive transfer of CD4+ and CD8+ T cells revealed that immune memory, after tumor rechallenging, was driven by CD4+ T cells. Collectively, these results illustrate the ability of MBTA therapy to activate both parts of the immune system and render a systemic antitumor response against non-treated metastases. We believe that our results could lead to the use of MBTA therapy in patients with aggressive, metastatic lesions. Reference: Caisova et al., Cancers (Basel), 2019. 11(5).

We examined the efficacy of selective inhibition of cyclin-dependent kinase 5 (CDK5) in glioblastoma by TP5. We analyzed its impact in vitro on CDK5 expression and activity, cell survival, apoptosis and cell cycle. DNA damage was analyzed using the expression of &gamma, H2A.X and phosphorylated ATM. Its tolerance and efficacy were assessed on in vivo xenograft mouse models. We showed that TP5 decreased the activity but not the expression of CDK5 and p35. TP5 alone impaired cell viability and colony formation of glioblastoma cell lines and induced apoptosis. TP5 increased DNA damage by inhibiting the phosphorylation of ATM, leading to G1 arrest. Whereas CDK5 activity is increased by DNA-damaging agents such as temozolomide and irradiation, TP5 was synergistic with either temozolomide or irradiation due to an accumulation of DNA damage. Concomitant use of TP5 and either temozolomide or irradiation reduced the phosphorylation of ATM, increased DNA damage, and inhibited the G2/M arrest induced by temozolomide or irradiation. TP5 alone suppressed the tumor growth of orthotopic glioblastoma mouse model. The treatment was well tolerated. Finally, alone or in association with irradiation or temozolomide, TP5 prolonged mouse survival. TP5 alone or in association with temozolomide and radiotherapy is a promising therapeutic option for glioblastoma.

The genetic basis and corresponding clinical relevance of prolactinomas remain poorly understood. Here, we perform whole genome sequencing (WGS) on 21 patients with prolactinomas to detect somatic mutations and then validate the mutations with digital polymerase chain reaction (PCR) analysis of tissue samples from 227 prolactinomas. We identify the same hotspot somatic mutation in splicing factor 3 subunit B1 (SF3B1R625H) in 19.8% of prolactinomas. These patients with mutant prolactinomas display higher prolactin (PRL) levels (p = 0.02) and shorter progression-free survival (PFS) (p = 0.02) compared to patients without the mutation. Moreover, we identify that the SF3B1R625H mutation causes aberrant splicing of estrogen related receptor gamma (ESRRG), which results in stronger binding of pituitary-specific positive transcription factor 1 (Pit-1), leading to excessive PRL secretion. Thus our study validates an important mutation and elucidates a potential mechanism underlying the pathogenesis of prolactinomas that may lead to the development of targeted therapeutics., The genetic basis of prolactinomas remains poorly understood. Here, the authors find a recurrent hotspot somatic mutation in the splicing factor 3 subunit B1 (SF3B1R625H) in prolactinomas, and show that this mutation causes aberrant splicing of ESRRG mRNA leading to up-regulation of prolactin.

PURPOSE: Glioblastoma (GBM) carries a dismal prognosis despite standard multimodal treatment with surgery, chemotherapy and radiation. Immune checkpoint inhibitors, such as PD1 blockade, for treatment of GBM failed to show clinical benefit. Rational combination strategies to overcome resistance of GBM to checkpoint monotherapy are needed to extend the promise of immunotherapy to GBM management. Emerging evidence suggests that protein phosphatase 2A (PP2A) plays a critical role in the signal transduction pathways of both adaptive and innate immune cells and that inhibition of PP2A could enhance cancer immunity. We investigated the use of a PP2A inhibitor, LB-100, to enhance antitumor efficacy of PD1 blockade in a syngeneic glioma model. METHODS: C57BL/6 mice were implanted with murine glioma cell line GL261-luc or GL261-WT and randomized into 4 treatment arms: (i) control, (ii) LB-100, (iii) PD1 blockade and (iv) combination. Survival was assessed and detailed profiling of tumor infiltrating leukocytes was performed. RESULTS: Dual PP2A and PD1 blockade significantly improved survival compared with monotherapy alone. Combination therapy resulted in complete regression of tumors in about 25% of mice. This effect was dependent on CD4 and CD8 T cells and cured mice established antigen-specific secondary protective immunity. Analysis of tumor lymphocytes demonstrated enhanced CD8 infiltration and effector function. CONCLUSION: This is the first preclinical investigation of the effect of combining PP2A inhibition with PD1 blockade for GBM. This novel combination provided effective tumor immunotherapy and long-term survival in our animal GBM model.

Triple-negative breast cancer (TNBC) does not respond to widely used targeted/endocrine therapies because of the absence of progesterone and estrogen receptors and HER2 amplification. It has been shown that the majority of TNBC cells are highly sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, but the development of TRAIL resistance limits its efficacy. We previously found that protein phosphatase 2A (PP2A) plays an important role in TRAIL resistance. In this study, we evaluated the effects of PP2A inhibition on cell death in TRAIL-resistant TNBC cells. We found that the PP2A inhibitor LB-100 effectively inhibits the growth of a panel of TNBC cell lines including lines that are intrinsically resistant to TRAIL. Using two TRAIL-resistant cell lines generated from TRAIL-sensitive parental cells (MDA231 and SUM159), we found that both TRAIL-sensitive and -resistant cell lines are equally sensitive to LB-100. We also found that LB-100 sensitizes TNBC cells to clinically used chemotherapeutical agents, including paclitaxel and cisplatin. Importantly, we found that LB-100 effectively inhibits the growth of MDA468 tumors in mice in vivo without apparent toxicity. Collectively, these data suggest that pharmacological inhibition of PP2A activity could be a novel therapeutic strategy for treating patients with TNBC in a clinical setting.

Chimeric antigen receptor (CAR)-engineered T cells represent a promising modality for treating glioblastoma. Recently, we demonstrated that CAR-T cells targeting carbonic anhydrase IX (CAIX), a protein involved in HIF-1a hypoxic signaling, is a promising CAR-T cell target in an intracranial murine glioblastoma model. Anti-CAIX CAR-T cell therapy is limited by its suboptimal activation within the tumor microenvironment. LB-100, a small molecular inhibitor of protein phosphatase 2A (PP2A), has been shown to enhance T cell anti-tumor activity through activation of the mTOR signaling pathway. Herein, we investigated if a treatment strategy consisting of a combination of LB-100 and anti-CAIX CAR-T cell therapy produced a synergistic anti-tumor effect. Our studies demonstrate that LB-100 enhanced anti-CAIX CAR-T cell treatment efficacy in vitro and in vivo. Our findings demonstrate the role of LB-100 in augmenting the cytotoxic activity of anti-CAIX CAR-T cells and underscore the synergistic therapeutic potential of applying combination LB-100 and CAR-T Cell therapy to other solid tumors.

Background/Aims: Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase that mediates cell cycle regulation and metabolism. Mounting evidence has indicated that PP2A inhibition exhibits considerable anticancer potency in multiple types of human cancers. However, the efficacy of PP2A inhibition remains unexplored in mucoepidermoid carcinoma (MEC), especially in locally advanced and metastatic cases with limited systemic treatment. In this study, we demonstrated the therapeutic potency of LB100 in mucoepidermoid carcinoma. Methods: In this study, the expression of PP2A was evaluated using immunohistochemical (IHC) staining. The effects associated with LB100 alone and in combination with cisplatin for the treatment of mucoepidermoid carcinoma were investigated both in vitro, regarding metabolism, proliferation, and migration, and in vivo in a mucoepidermoid carcinoma xenograft model. In addition, with LB100 treatment and in response to an insulin stimulus, the expression levels and phosphorylation levels of targets in the PI3K-AKT pathway were determined using western blot analysis and immunoblotting. Results: The expression of protein phosphatase 2A was significantly upregulated in the clinical specimens of high-grade MECs compared with those of low-/medium-grade MECs and normal controls. In this article, we report that a small molecule PP2A inhibitor, LB100, decreased cellular viability and glycolytic activity and induced G2/M cell cycle arrest. Importantly, LB100 enhanced the efficacy of cisplatin in mucoepidermoid carcinoma cells both in vitro and in vivo. PP2A inhibition by LB100 increased the phosphorylation of insulin receptor substrate 1(IRS-1) on serine residues, downregulated the expression of phosphatidylinositol 3-kinase (PI3K) p110 alpha subunit and dephosphorylated AKT at Ser473 and Thr308 in mucoepidermoid carcinoma cells in response to insulin stimulus. Conclusion: These results highlight the translational potential of PP2A inhibition to synergize with cisplatin in mucoepidermoid carcinoma treatment.

INTRODUCTION Glioblastoma (GBM) is the most common malignant primary brain tumor and has a heterogeneous tumor cell population. The median survival of GBM patients is less than two years with current multi-model therapy of maximal surgical resection followed by chemotherapy, radiation and tumor treating fields. Protein Arginine Methyltransferase 5 (PRMT5) regulates cellular functions through symmetric di-methylation of arginine residues of histone and non-histone proteins. Our recent findings show that PRMT5 is overexpressed in GBM; its inhibition causes apoptosis and senescence of mature and immature GBM tumor cells respectively. Protein Phosphatase 2A (PP2A), a serine-threonine phosphatase, is associated with senescent tumor cells that contribute to therapy resistance; LB100 is a first-in-class small molecule inhibitor of PP2A that can sensitize tumor cells that are chemo- and radiation-therapy resistant. In this study, using patient-derived primary GBM neurospheres (GBMNS), we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. METHODS Patient-derived primary GBM neurospheres transfected with PRMT5 target-specific siRNA were treated with LB100 and subjected to in vitro assays such as proliferation assay, cell cycle analysis, cytotoxicity assay, PP2A activity and western blot. RESULTS LB100 treatment significantly reduced the viability of PRMT5-depleted GBM cells as compared to PRMT5 intact cells. LB100 caused G1 cell cycle arrest through inactivation of Rb protein; this effect is further enhanced in combination with PRMT5-depletion. Combination therapy also increased the expression of phospho-MLKL; Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100 indicating that necroptosis caused the enhanced cytotoxicity in combination therapy. We also found that PRMT5-depletion increased the PP2A activity in GBM neurospheres. CONCLUSION Combining the PRMT5 inhibition and PP2A inhibition produced greater antitumor effects than with PRMT5 inhibition alone.

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src–cell membrane association–dissociation and catalytic activation–inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825–38. ©2018 AACR.

PURPOSE: Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors of neural crest origin. Germline or somatic mutations of numerous genes have been implicated in the pathogenesis of PPGLs, including the isocitrate dehydrogenase 1 (IDH1) gene and alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene. Although concurrent IDH1 and ATRX mutations are frequently seen in gliomas, they have never been reported together in PPGLs. The aim of this study was to characterize one paraganglioma with concurrent IDH1 and ATRX mutations identified by whole exome sequencing. METHODS: Leukocyte and tumor DNA were used for whole exome sequencing and Sanger sequencing. 2-hydroxyglurarate level and the global DNA methylation status in the tumor were measured. ATRX’s cDNA transcripts were analyzed. Tyrosine hydroxylase (TH), HIF1α and ATRX staining, as well as telomere-specific FISH was also performed. RESULTS: The presence of a somatic IDH1 (c.394C>T, p.R132C) mutation and a concurrent somatic ATRX splicing mutation (c.4318–2A>G) in the current case was confirmed. Dramatic accumulation of 2-hydroxyglutarate was detected in the paraganglioma without the global DNA hypermethylation, and pseudohypoxia was also activated. Importantly, immunohistochemistry revealed negative TH staining in the tumor and the first exon region of TH gene was hypermethylated resulting in normal plasma metanephrines. The splicing ATRX mutation resulted in two transcripts, causing frameshifts. Immunohistochemistry revealed scarce ATRX staining in the tumor. Alternative lengthening of telomeres (ALT) was detected by FISH. CONCLUSIONS: This case represents the first concurrence of IDH1 and ATRX mutations in PPGLs. Although relatively rare, a somatic R132C mutation of IDH1 might play a role in a small subset of sporadic PPGLs.

The myelodysplastic syndromes (MDS) are a group of hematologic disorders characterized by the presence of somatically mutated hematopoietic stem cells (HSCs) that increase the risk of progression to secondary acute myeloid leukemia (sAML). Mutations in isocitrate dehydrogenase (IDHmut) are thought to correlate with the increased production of the oncogenic protein 2-hydroxyglutarate (2-HG) in AML. The aim of this study was to examine whether serum 2-HG has utility as a prognostic biomarker, and whether elevated 2-HG levels are predictive of IDH mutations in patients with MDS. Genetic profiling was utilized to determine the genetic composition of a large cohort of MDS patients, including the presence or absence of IDH1 or IDH2 mutations (n = 281). Serum 2-HG levels were detected by liquid chromatography–tandem mass spectrometry. In the current study of MDS patients, elevated serum 2-HG levels were predictive of inferior overall- and leukemia-free survival irrespective of IPSS risk grouping. Higher serum 2-HG levels predicted the presence of IDH mutations. IDH2mut patients had a higher risk of leukemic transformation. The co-occurrence of DNMT3A or SRSF2 mutations was found to be increased in IDH2mut patients. IDH2 mutations were associated with significantly worse OS and LFS amongst patients with low-risk MDS by IPSS grouping. The noted predictive value of serum 2-HG levels and IDH2 mutations on OS and LFS support the use of biomarkers and/or underlying cytogenetics in novel prognostic scoring systems for MDS.

Oral mucosal melanoma (OMM) is a rare and aggressive subtype of melanoma with little known about its pathogenesis or carcinogenesis. We therefore performed whole-exome sequencing (WES) on 19 matched OMM tumor/normal pairs in order to gain insight into potential genetic drivers of tumor formation. For the first time, we describe the comprehensive mutational profile of OMM. Our data suggest that the genetic background of OMM differs from those of other melanoma subtypes. We identified recurrent mutations involving KIT, POLE, PTPRD, PTCHD2, and DMXL2. Notably, copy number analysis revealed recurrently amplified regions of 12q14 (57.9%, containing CDK4) and 5p15 (47.4%, containing TERT). CNV analysis in a separate cohort of 15 samples validated the frequent CNV in CDK4 and TERT. We also observed that the melanocyte development and pigmentation signaling pathway is frequently altered in OMM. Furthermore, our data suggest several altered genes that may be amenable for targeted therapy. We identified one patient with metastatic OMM in our cohort who was identified to harbor a targetable KIT mutation using our WES results. This patient was able to achieve complete remission following implementation of KIT-targeted therapy. These findings provide further insight into the genetic underpinnings of OMM development and suggest that patients with OMM may benefit from WES analysis to identify potential targetable genetic mutations. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tumor-associated macrophages (TAMs) are predominantly M2 phenotype in solid cancers including hepatocellular carcinoma (HCC). Though differentiation of M2 macrophages has been recently linked to fatty acid oxidation (FAO), whether FAO plays a role in functional maintenance of M2 macrophages is still unclear. Here, we used an in vitro model to mimic TAM-HCC interaction in tumor microenvironment. We found that M2 monocyte-derived macrophages (MDMs) enhanced the proliferation, migration, and invasion of HCC cells through an FAO-dependent way. Further investigations identified that IL-1β mediated the pro-migratory effect of M2 MDM. Using etomoxir and siRNA to inhibit FAO and palmitate to enhance FAO, we showed that FAO was responsible for the up-regulated secretion of IL-1β and, thus, the pro-migratory effect in M2 MDMs. In addition, we proved that IL-1β induction was reactive oxygen species and NLRP3-dependent. Our study demonstrates that FAO plays a key role in functional human M2 macrophages by enhancing IL-1β secretion to promote HCC cell migration. These findings provide evidence for different dependency of energy sources in macrophages with distinct phenotypes and functions, and suggest a novel strategy to treat HCC by reprogramming cell metabolism or modulating tumor microenvironment.

Atypical teratoid rhabdoid tumor (ATRT) is a pediatric brain tumor with a high mortality rate characterized by mutations in/ deletions of SWI/SNF matrix-associated actin-dependent regulator of chromatin sub-family B member 1 (SMARCB1). We previously showed that loss of SMARCB1 causes up-regulation and release of HML-2 subfamily of human endogenous retrovirus K envelope protein (HML-2 ENV), resulting in maintenance of pluripotency. Here, we investigated intracellular trafficking and release of HML-2 ENV. Further, we demonstrate two potential therapeutic strategies to decrease intracellular HML-2 ENV: 1) inhibition of calcium influx by ouabain, a cardiac glycoside toxic to neural stem cells, and 2) targeted inhibition of cyclin-dependent kinase 5 (CDK5), which is restricted to neurons by p35, its activator protein, by TP5. ATRT cell lines and tumor tissue obtained from patients were confirmed for SMARCB1 loss and increased HML-2 ENV. Cell viability and intracellular HML-2 ENV concentration were measured after treatment with ouabain and TP5 (CDK5 antagonist). We evaluated the calcium-mediated effect of ouabain on HML-2 intracellular concentration by treating the cells with ouabain, the calcium chelators calcimycin and EGTA, and calpeptin, a calpain inhibitor, which activates CDK5, and measuring HML-2 ENV and p35. We evaluated HML-2 ENV for a CDK5 consensus phosphorylation site and performed co-immunoprecipitation to evaluate direct interaction. We evaluated activity of CDK5 in ATRT cell lines by autoradiography. Both Ouabain and TP5 caused a decrease in cell viability in a dose-dependent manner. Further, ouabain treatment decreased HML-2 ENV intracellular concentration. We found that HML-2 ENV contains a consensus phosphorylation site for CDK5. We discovered that HML-2 ENV was bound to CDK5. We established that ATRT cell lines had hyperactive CDK5. Finally, we established that the effect of ouabain on HML-2 ENV was due to indirect inhibition of calcium-mediated activation of calpain and thus CDK5.

Background Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Methods Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. Results PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Conclusions Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma.

// Chao Zhang 1,2,* , Chunzhang Yang 3,* , Michael J. Feldman 4 , Herui Wang 4 , Ying Pang 2 , Dominic M. Maggio 4 , Dongwang Zhu 4 , Cody L. Nesvick 4 , Pauline Dmitriev 4 , Petra Bullova 2,5 , Prashant Chittiboina 4 , Roscoe O. Brady 4 , Karel Pacak 2 and Zhengping Zhuang 4 1 Department of Orthopedics, Xinqiao Hospital, The Third Military Medical University, Chongqing, China 2 Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA 3 Neuro-Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA 4 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA 5 Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovakia * These authors have contributed equally to this work Correspondence to: Karel Pacak, email: // Zhengping Zhuang, email: // Keywords : HDACi, Hsp90, SAHA, HIF, hypoxia Received : December 14, 2016 Accepted : April 10, 2017 Published : May 23, 2017 Abstract Histone deacetylase inhibitors (HDACis) are a potent class of tumor-suppressive agents traditionally believed to exert their effects through loosening tightly-wound chromatin resulting in de-inhibition of various tumor suppressive genes. Recent literature however has shown altered intratumoral hypoxia signaling with HDACi administration not attributable to changes in chromatin structure. We sought to determine the precise mechanism of HDACi-mediated hypoxia signaling attenuation using vorinostat (SAHA), an FDA-approved class I/IIb/IV HDACi. Through an in-vitro and in-vivo approach utilizing cell lines for hepatocellular carcinoma (HCC), osteosarcoma (OS), and glioblastoma (GBM), we demonstrate that SAHA potently inhibits HIF-a nuclear translocation via direct acetylation of its associated chaperone, heat shock protein 90 (Hsp90). In the presence of SAHA we found elevated levels of acetyl-Hsp90, decreased interaction between acetyl-Hsp90 and HIF-a, decreased nuclear/cytoplasmic HIF-α expression, absent HIF-α association with its nuclear karyopharyin Importin, and markedly decreased HIF-a transcriptional activity. These changes were associated with downregulation of downstream hypoxia molecules such as endothelin 1, erythropoietin, glucose transporter 1, and vascular endothelial growth factor. Findings were replicated in an in-vivo Hep3B HRE-Luc expressing xenograft, and were associated with significant decreases in xenograft tumor size. Altogether, this study highlights a novel mechanism of action of an important class of chemotherapeutic.

Immunotherapy based on activation of innate immunity has been tested in syngeneic mouse tumor models via intratumoral administration of the following components: phagocytosis-stimulating ligands (Mannan-BAM), toll-like receptor (TLR) agonists, and immunostimulant anti-CD40 antibody (abbreviated as MBTA). In this study, syngeneic colon carcinoma (CT26) and glioma (GL261) models were established to assess MBTA’s efficacy in generating immune responses against distal metastatic lesions and CNS tumors. Additionally, we investigated if therapeutic delivery of MBTA could be optimized beyond intratumoral delivery. In the colon carcinoma model, intratumoral injection of MBTA significantly reduced all metastatic CT26 tumor growth rate and induced complete remission (CR) in 33% (3/9) of treated animals. In the glioma model, subcutaneous injection of GL261 cells incubated with MBTA resulted in the complete regression of intracranial gliomas in 87.5% (7/8) of treated animals. Therapeutic effect of MBTA was abrogated in CD4+ and CD8+ lymphocyte depleted mice. Tumor infiltrating leukocyte analyses demonstrated significantly increased CD8+ cytotoxic T-lymphocytes (CTL) in metastatic tumors with higher percentages of TNFα and IFNγ positive cells. Further assessments with MHC I tetramers revealed significantly increased CT26-associated peptide (AH1) specific CTLs in the blood of MBTA treated animals. All animals that achieved complete remission in the colon carcinoma model resisted subsequent peripheral and intracranial challenges with CT26 cells, confirming the induction of immunological memory against CT26 tumors. Collectively, our investigation demonstrates that MBTA can effectively induce a tumor-specific adaptive immune response that can target tumors located in the periphery and within the CNS.

Advances in the understanding of growth hormone-producing adenomas (GHomas) are ongoing, but current therapy is limited by moderate and variable efficacy and in need of life-long treatment. In this study, the molecular signaling pathway related to GHoma was investigated by proteomics and transcriptomics. The differentially expressed proteins and genes were significantly enriched in Extracellular Matrix-Receptor Interactions, Notch Signaling, Basal Cell Carcinoma Signaling, JAK-STAT3, Wnt Signaling, and Glioblastoma Multiforme Signaling by Ingenuity Pathway Analysis. Furthermore, the Notch2/Delta-like canonical Notch ligand (DLL) signaling pathway was identified to be associated with tumorigenesis and invasiveness of GHoma. In 76 patients, Notch2 and DLL3 were upregulated in invasive compared to those in non-invasive GHoma (p < 0.05). Disease-free survival was significantly longer in patients with low, compared with high, DLL3 expression (p = 0.027). Notch 2 knockdown inhibited cell migration in both GH3 cells and primary GHoma cells, along with downregulation of the mRNA expression of related genes. DAPT, a γ-secretase inhibitor, inhibited tumor growth and invasion in vivo and in vitro and suppressed the release of growth hormone in primary GHoma cells. The involvement of Notch2/DLL3 signaling in GHoma progression warrants additional study of Notch inhibitor, DAPT, as a potential GHoma treatment. Importance of the Study Current treatments of GH adenomas (GHomas) are limited by their moderate and variable efficacy and in need of life-long treatment. We found that the Notch2/Delta-like Notch ligand 3 (DLL3) signaling pathway was active in GHoma tumorigenesis, progression, and invasion. The γ-secretase inhibitor DAPT is of potential use in GHoma treatment targeting Notch signaling.

Therapeutic options for metastatic pheochromocytoma/paraganglioma (PHEO/PGL) are limited. Here, we tested an immunotherapeutic approach based on intratumoral injections of mannan-BAM with toll-like receptor ligands into subcutaneous PHEO in a mouse model. This therapy elicited a strong innate immunity-mediated antitumor response and resulted in a significantly lower PHEO volume compared to the phosphate buffered saline (PBS)-treated group and in a significant improvement in mice survival. The cytotoxic effect of neutrophils, as innate immune cells predominantly infiltrating treated tumors, was verified in vitro. Moreover, the combination of mannan-BAM and toll-like receptor ligands with agonistic anti-CD40 was associated with increased mice survival. Subsequent tumor re-challenge also supported adaptive immunity activation, reflected primarily by long-term tumor-specific memory. These results were further verified in metastatic PHEO, where the intratumoral injections of mannan-BAM, toll-like receptor ligands, and anti-CD40 into subcutaneous tumors resulted in significantly less intense bioluminescence signals of liver metastatic lesions induced by tail vein injection compared to the PBS-treated group. Subsequent experiments focusing on the depletion of T cell subpopulations confirmed the crucial role of CD8+ T cells in inhibition of bioluminescence signal intensity of liver metastatic lesions. These data call for a new therapeutic approach in patients with metastatic PHEO/PGL using immunotherapy that initially activates innate immunity followed by an adaptive immune response.

We previously identified a novel syndrome in patients characterized by paraganglioma, somatostatinoma, and polycythemia. In these patients, polycythemia occurs long before any tumor develops, and tumor removal only partially corrects polycythemia, with recurrence occurring shortly after surgery. Genetic mosaicism of gain-of-function mutations of the EPAS1 gene (encoding HIF2&alpha, ) located in the oxygen degradation domain (ODD), typically p.530&ndash, 532, was shown as the etiology of this syndrome. The aim of the present investigation was to demonstrate that these mutations are necessary and sufficient for the development of the symptoms. We developed transgenic mice with a gain-of-function Epas1A529V mutation (corresponding to human EPAS1A530V), which demonstrated elevated levels of erythropoietin and polycythemia, a decreased urinary metanephrine-to-normetanephrine ratio, and increased expression of somatostatin in the ampullary region of duodenum. Further, inhibition of HIF2&alpha, with its specific inhibitor PT2385 significantly reduced erythropoietin levels in the mutant mice. However, polycythemia persisted after PT2385 treatment, suggesting an alternative erythropoietin-independent mechanism of polycythemia. These findings demonstrate the vital roles of EPAS1 mutations in the syndrome development and the great potential of the Epas1A529V animal model for further pathogenesis and therapeutics studies.

Background The bromodomain and extra-terminal domain (BET) inhibitor is a type of anti-tumor agent, currently being evaluated in phase I and II clinical trials for cancer therapy. It can decrease MYC expression levels and cause effective anti-tumor effects in diverse human cancers. However, its cytotoxic effect and related mechanisms of drug resistance are poorly understood in hepatocellular carcinomas (HCC). Here, we investigated the anti-tumor effects of BET inhibitor on HCC and the molecular mechanisms involved in its associated drug resistance. Methods We assessed the cytotoxicity of BET inhibitor on HCC cells compared with sorafenib by cell viability assay, metastasis assay and reproduced the anti-tumor effect in xenograft mouse model. In addition, the molecular mechanisms involved in drug resistance on JQ1-resistant HCC cells were revealed by western blotting, qRT-PCR, whole exome-sequencing and gene-editing technology. Finally, with specific inhibition of EGFR or ERK activity by interference RNAs or inhibitors, the efficacy of the synergistic treatment was investigated using cell viability assay, colony formation, apoptosis and xenograft mouse model. Results We found that JQ1, a commonly used BET bromo-domain inhibitor, offered a better anti-tumor response than sorafenib in MYC-positive HCC cells by inducing apoptosis in vitro and in vivo. Unlike sorafenib, JQ1 treatment significantly impaired mitochondrial respiration and glycolysis in HCC cells. Importantly, we revealed that MAPK activation by a previously undescribed activating mutation of EGFR-I645L, was critical for JQ1 sensitivity through stabilizing oncogenic MYC protein in JQ1-resistant HCC cells. Inhibition of either EGFR or ERK activity overcame the JQ1 resistance and significantly decreased MYC protein level in vitro and in vivo. Conclusion Since MYC amplification is frequently identified in HCC, co-occurring with EGFR amplification, our findings suggest that targeting EGFR signaling might be essential for JQ1 therapy in advanced HCC. Electronic supplementary material The online version of this article (10.1186/s13046-019-1082-6) contains supplementary material, which is available to authorized users.

BACKGROUND: Increasing evidence suggests a role of increased expression of CDK5 in cancer progression. Here, we examined the efficacy of selective inhibition of CDK5 in glioblastoma, the most frequent and aggressive primary brain tumor in adults. TP5 is a small peptide developed by National Institutes of Health which selectively inhibits aberrant and hyperactive CDK5/p25 while preserving the physiological CDK5/p35 functions. This peptide was modified to facilitate passage through blood brain barrier. METHODS & RESULTS: We demonstrated that TP5 decreased the activity but not the expression of CDK5 and p35. TP5 alone, but not the scrambled peptide, impacted cell viability and colony formation of glioblastoma cell lines and increased early and late apoptosis. Analyzing the pH2A.X expression by Immunofluorescence and Western Blot, we observed that TP5 increased the DNA damage in a dose dependent manner. TP5 impaired the DNA repair by reducing the G2 phase and decreased the phosphorylation of ATM. Whereas CDK5 activity is increased by DNA-damaging agents such as temozolomide (TMZ) and irradiation (IR), we observed that the addition of TP5 to either TMZ or IR was synergistic on colony formation due to an accumulation of DNA damages. Concomitant use of TP5 and either TMZ or IR increased the pH2A.X foci number and expression, inhibited the G2 arrest induced by these therapies and the phosphorylation of ATM. TP5 alone (300 mg/kg every 2 days) decreased the tumor volume of orthotopic glioblastoma mouse-model in a safety and dose-escalation experiment (4 mice per group: control, scrambled peptide, 100mg/kg, 300mg/kg). The treatment was well tolerated as the mouse weights were stable during the treatment and no hematologic, renal, pancreatic or liver function toxicities were observed (blood test). CONCLUSIONS: TP5 is a promising therapy for glioblastoma patients alone or in association with temozolomide and radiotherapy targeting a unique aspect of tumor biology.

BACKGROUND: Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor treating fields, median survival of Glioblastoma (GBM) patients is less than two years. Protein Arginine Methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence of immature GBM tumor cells. LB100, first-in-class small molecule inhibitor of Protein Phosphatase 2A (PP2A) can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. Methods: Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. Results: We found that PRMT5-depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5 intact GBMNS. LB100 enhanced the G1 cell cycle arrest induced by PRMT5-depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged mice survival, while LB100 treatment alone had no survival benefit. Conclusion: Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

Metabolomics has shown significant potential in identifying small molecules specific to tumor phenotypes. In this study we analyzed resected tissue metabolites using capillary electrophoresis-mass spectrometry and found that tissue hypotaurine levels strongly and positively correlated with glioma grade. In vitro studies were conducted to show that hypotaurine activates hypoxia signaling through the competitive inhibition of prolyl hydroxylase domain-2. This leads to the activation of hypoxia signaling as well as to the enhancement of glioma cell proliferation and invasion. In contrast, taurine, the oxidation metabolite of hypotaurine, decreased intracellular hypotaurine and resulted in glioma cell growth arrest. Lastly, a glioblastoma xenograft mice model was supplemented with taurine feed and exhibited impaired tumor growth. Taken together, these findings suggest that hypotaurine is an aberrantly produced oncometabolite, mediating tumor molecular pathophysiology and progression. The hypotaurine metabolic pathway may provide a potentially new target for glioblastoma diagnosis and therapy.

The protein phosphatase 2A (PP2A) inhibitor, LB100, has been shown in pre-clinical studies to be an effective chemo- and radio-sensitizer for treatment of various cancers. We investigated effects associated with LB100 treatment alone and in combination with cisplatin for medulloblastoma (MB) in vitro and in vivo in an intracranial xenograft model. We demonstrated that LB100 had a potent effect on MB cells. By itself, LB100 inhibited proliferation and induced significant apoptosis in a range of pediatric MB cell lines. It also attenuated MB cell migration, a pre-requirement for invasion. When used in combination, LB100 enhanced cisplatin-mediated cytotoxic effects. Cell viability in the presence of 1 uM cisplatin alone was 61% (DAOY), 100% (D341), and 58% (D283), but decreased with the addition of 2 μM of LB100 to 26% (DAOY), 67% (D341), and 27% (D283), (p < 0.005). LB100 suppressed phosphorylation of the STAT3 protein and several STAT3 downstream targets. Also, LB100 directly increased cisplatin uptake and overcame cisplatin-resistance in vitro. Finally, LB100 exhibited potent in vivo anti-neoplastic activity in combination with cisplatin in an intracranial xenograft model.

Emerging evidence is demonstrating the extent of T-cell infiltration within the tumor microenvironment has favorable prognostic and therapeutic implications. Hence, immunotherapeutic strategies that augment the T-cell signature of tumors hold promising therapeutic potential. Recently, immunotherapy based on intratumoral injection of mannan-BAM, toll-like receptor ligands and anti-CD40 antibody (MBTA) demonstrated promising potential to modulate the immune phenotype of injected tumors. The strategy promotes the phagocytosis of tumor cells to facilitate the recognition of tumor antigens and induce a tumor-specific adaptive immune response. Using a syngeneic colon carcinoma model, we demonstrate MBTA’s potential to augment CD8(+) T-cell tumor infiltrate when administered intratumorally or subcutaneously as part of a whole tumor cell vaccine. Both immunotherapeutic strategies proved effective at controlling tumor growth, prolonged survival and induced immunological memory against the parental cell line. Collectively, our investigation demonstrates MBTA’s potential to trigger a potent anti-tumor immune response.

BACKGROUND: Somatic mutations in hypoxia-inducible factor 2α (HIF2A) is associated with polycythemia-paraganglioma syndrome. Specifically, the classic presentation of female patients with recurrent paragangliomas (PGLs), polycythemia (at birth or in early childhood), and duodenal somatostatinomas has been described. Studies have demonstrated that somatic HIF2A mutations occur as postzygotic events and some to be associated with somatic mosaicism affecting hematopoietic and other tissue precursors. This phenomenon could explain the development of early onset of polycythemia in the absence of erythropoietin secreting tumors. METHODS: Correlation analysis was performed between mosaicism of HIF2A mutant patients and clinical presentations. RESULTS: Somatic HIF2A mutations (p.A530V, p.P531S and p.D539N) were identified in DNA extracted from PGLs of three patients. No somatic mosaicism was detected through deep sequencing of blood genomic DNA. Compared to classic syndrome, both polycythemia and PGL in all three patients developed at an advanced age with polycythemia at 30, 30, and 17 and PGLs at age 34, 30, and 55 years, respectively. Somatostatinomas were not detected and two patients had ophthalmic findings. The biochemical phenotype in all three patients was noradrenergic with (18)F-fluorodopa PET/CT as the most sensitive imaging modality. All patients demonstrated multiplicity and none developed metastatic disease. CONCLUSIONS: These findings suggest that newer techniques need to be developed to detect germline or somatic mosaicism in patients with this syndrome. Absence of HIF2A mosaicism in patients with somatic HIF2A mutations supports association with late onset of the disease, milder clinical phenotype and an improved prognosis compared to patients with HIF2A mosaicism.

Mounting evidence suggests that inhibition of protein phosphatase-2A (PP2A), a serine/threonine phosphatase, could enhance anticancer immunity. However, drugs targeting PP2A are not currently available. Here, we report that a PP2A inhibitor, LB-100, when combined with anti-PD-1 (aPD-1) blockade can synergistically elicit a durable immune-mediated antitumor response in a murine CT26 colon cancer model. This effect is T-cell dependent, leading to regression of a significant proportion of tumors. Analysis of tumor lymphocytes demonstrates enhanced effector T-cell and reduced suppressive regulatory T-cell infiltration. Clearance of tumor establishes antigen-specific secondary protective immunity. A synergistic effect of LB-100 and aPD-1 blockade is also observed in B16 melanoma model. In addition, LB-100 activates the mTORC1 signaling pathway resulting in decreased differentiation of naive CD4 cells into regulatory T cells. There is also increased expression of Th1 and decreased expression of Th2 cytokines. These data highlight the translational potential of PP2A inhibition in combination with checkpoint inhibition., Protein phosphatase 2A (PP2A) has been proposed as a target for cancer immunotherapy. Here the authors show that pharmacological inhibition of PP2A with a clinically-relevant inhibitor enhances response to immune checkpoint blockade in pre-clinical models of cancer, resulting in long lasting immunity.