HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity.

Proteasomes degrade intracellular proteins to generate antigenic peptides that are recognized by the adaptive immune system and promote anticancer immunity. However, tumors subvert the antigen presentation machinery to escape immunosurveillance. We hypothesized that proteasome activation could concomitantly increase antigen abundance and diversity in multiple myeloma cells. High-throughput screens revealed that histone deacetylase 6 (HDAC6) inhibitors activated proteasomes to unmask neoantigens and amplify the tumor-specific antigenic landscape. Treatment of patient CD138+ cells with HDAC6 inhibitors significantly promoted the antimyeloma activity of autologous CD8+ T cells. Pharmacologic blockade and genetic ablation of the HDAC6 ubiquitin-binding domain released HR23B, which shuttles ubiquitinylated cargo to proteasomes, while silencing HDAC6 or HR23B in multiple myeloma cells abolished the effect of HDAC6 inhibitors on proteasomes, antigen presentation, and T-cell cytotoxicity. Taken together, our results demonstrate the paradigm-shifting translational impact of proteasome activators to expand the myeloma immunopeptidome and have revealed novel, actionable antigenic targets for T cell-directed immunotherapy. SIGNIFICANCE: The elimination of therapy-resistant tumor cells remains a major challenge in the treatment of multiple myeloma. Our study identifies and functionally validates agents that amplify MHC class I-presented antigens and pave the way for the development of proteasome activators as immune adjuvants to enhance immunotherapeutic responses in patients with multiple myeloma.

Targeting Proteasomes and the MHC Class I Antigen Presentation Machinery to Treat Cancer, Infections and Age-Related Diseases.

The majority of T-cell responses involve proteasome-dependent protein degradation and the downstream presentation of oligopeptide products complexed with major histocompatibility complex (MHC) class I (MHC-I) molecules to peptide-restricted CD8+ T-cells. However, evasion of host immunity is a cancer hallmark that is achieved by disruption of host antigen processing and presentation machinery (APM). Consequently, mechanisms of immune evasion promote cancer growth and survival as well as de novo and acquired resistance to immunotherapy. A multitude of cell signaling pathways modulate the APM and MHC-I-dependent antigen presentation. Pharmacologics that specifically target and modulate proteasome structure and activity represent a novel emerging strategy to improve the treatment of cancers and other diseases characterized by aberrant protein accumulation. FDA-approved pharmacologics that selectively activate proteasomes and/or immunoproteasomes can be repositioned to overcome the current bottlenecks that hinder drug development to enhance antigen presentation, modulate the immunopeptidome, and enhance the cytotoxic activity of endogenous or engineered T-cells. Strategies to enhance antigen presentation may also improve the antitumor activity of T-cell immunotherapies, checkpoint inhibitors, and cancer vaccines. Proteasomes represent actionable therapeutic targets to treat difficult-to-treat infectious processes and neurodegenerative diseases that are characterized by the unwanted accrual of insoluble, deleterious, and potentially toxic proteins. Taken together, we highlight the breadth and magnitude of the proteasome and the immense potential to amplify and unmask the immunopeptidomic landscape to improve the treatment of a spectrum of human diseases.

Current and Emerging Strategies to Treat Urothelial Carcinoma.

Urothelial cell carcinoma (UCC, bladder cancer, BC) remains a difficult-to-treat malignancy with a rising incidence worldwide. In the U.S., UCC is the sixth most incident neoplasm and ~90% of diagnoses are made in those >55 years of age; it is ~four times more commonly observed in men than women. The most important risk factor for developing BC is tobacco smoking, which accounts for ~50% of cases, followed by occupational exposure to aromatic amines and ionizing radiation. The standard of care for advanced UCC includes platinum-based chemotherapy and programmed cell death (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitors, administered as frontline, second-line, or maintenance therapy. UCC remains generally incurable and is associated with intrinsic and acquired drug and immune resistance. UCC is lethal in the metastatic state and characterized by genomic instability, high PD-L1 expression, DNA damage-response mutations, and a high tumor mutational burden. Although immune checkpoint inhibitors (ICIs) achieve long-term durable responses in other cancers, their ability to achieve similar results with metastatic UCC (mUCC) is not as well-defined. Here, we discuss therapies to improve UCC management and how comprehensive tumor profiling can identify actionable biomarkers and eventually fulfill the promise of precision medicine for UCC patients.

Vactosertib, a novel TGF-ß1 type I receptor kinase inhibitor, improves T-cell fitness: a single-arm, phase 1b trial in relapsed/refractory multiple myeloma.

Functional blockade of the transforming growth factor-beta (TGF-ß) signaling pathway improves the efficacy of cytotoxic and immunotherapies. We conducted a phase 1b study to determine the safety, efficacy, and maximal tolerated dose (200 mg po bid) of the potent, orally-available TGF-ß type I receptor kinase inhibitor vactosertib in relapsed and/or refractory multiple myeloma patients who had received ≥2 lines of chemoimmunotherapy. Vactosertib combined with pomalidomide was well-tolerated at all doses, had a manageable adverse event profile and induced durable responses with 80% progression-free survival (PFS-6) at 6 months, while pomalidomide alone historically achieved 20% PFS-6. Following treatment, the immunosuppressive marker PD-1 expression was reduced on patient CD8+ T-cells. Following ex vivo treatment, vactosertib decreased PD-1 expression on patient CD138+ cells, reduced PD-L1/PD-L2 on patient CD138+ cells and enhanced the anti-myeloma activity of autologous T-cells. Taken together, vactosertib is a safe immunotherapy that modulates the T-cell immunophenotype to reinvigorate T-cell fitness. Multiple myeloma (MM) is a genetically heterogeneous hematologic malignancy characterized by the excessive proliferation of clonal plasma cells (1, 2). MM remains mostly incurable but a small group of patients can achieve long-term remission (3). Treatment of MM presents unique challenges due to the complex molecular pathophysiology and genetic heterogeneity (4, 5). Given that MM is the second most common blood cancer characterized by cycles of remission and relapse, the development of new therapeutic modalities is crucial (6, 7). The prognosis for MM patients has improved substantially over the past two decades with the development of more effective therapeutics, e.g., proteasome inhibitors, and regimens that demonstrate greater anti-tumor activity (8-10). The management of RRMM represents a vital aspect of the overall care for patients with disease and a critical area of ongoing scientific and clinical research (10-12).

Therapeutics to harness the immune microenvironment in multiple myeloma.

Multiple myeloma (MM) remains an incurable, genetically heterogeneous disease characterized by the uncontrolled proliferation of transformed plasma cells nurtured within a permissive bone marrow (BM) microenvironment. Current therapies leverage the unique biology of MM cells and target the immune microenvironment that drives tumor growth and facilitates immune evasion. Proteasome inhibitors and immunomodulatory drugs were initially introduced to complement and have now supplanted cytotoxic chemotherapy as frontline anti-myeloma agents. Recently, monoclonal antibodies, bispecific antibodies, and chimeric antigen receptor T cells were developed to revamp the immune system to overcome immune suppression and improve patient responses. While current MM therapies have markedly extended patient survival, acquired drug resistance inevitably emerges and drives disease progression. The logical progression for the next generation of MM therapies would be to design and validate agents that prevent and/or overcome acquired resistance to immunotherapies. The complex BM microenvironment promotes resistance to both current anti-myeloma agents and emerging immunotherapies. Myeloma cells are intertwined with a complex BM immune microenvironment that contributes to the development of adaptive drug resistance. Here, we describe recently FDA-approved and investigational anti-myeloma agents that directly or indirectly target the BM microenvironment to prevent or overcome drug resistance. Synergistic effects of anti-myeloma agents may foster the development of rationally-designed drug cocktails that prevent BM-mediated resistance to immunotherapies.

Targeting Proteasomes in Cancer and Infectious Disease: A Parallel Strategy to Treat Malignancies and Microbes.

Essential core pathways of cellular biology are preserved throughout evolution, highlighting the importance of these pathways for both bacteria and human cancer cells alike. Cell viability requires a proper balance between protein synthesis and degradation in order to maintain integrity of the proteome. Proteasomes are highly intricate, tightly regulated multisubunit complexes that are critical to achieve protein homeostasis (proteostasis) through the selective degradation of misfolded, redundant and damaged proteins. Proteasomes function as the catalytic core of the ubiquitin-proteasome pathway (UPP) which regulates a myriad of essential processes including growth, survival, differentiation, drug resistance and apoptosis. Proteasomes recognize and degrade proteins that have been marked by covalently attached poly-ubiquitin chains. Deregulation of the UPP has emerged as an essential etiology of many prominent diseases, including cancer. Proteasome inhibitors selectively target cancer cells, including those resistant to chemotherapy, while sparing healthy cells. Proteasome inhibition has emerged as a transformative anti-myeloma strategy that has extended survival for certain patient populations from 3 to 8 years. The structural architecture and functional activity of proteasomes is conserved from Archaea to humans to support the concept that proteasomes are actionable targets that can be inhibited in pathogenic organisms to improve the treatment of infectious diseases. Proteasomes have an essential role during all stages of the parasite life cycle and features that distinguish proteasomes in pathogens from human forms have been revealed. Advancement of inhibitors that target Plasmodium and Mycobacterial proteasomes is a means to improve treatment of malaria and tuberculosis. In addition, PIs may also synergize with current frontline agents support as resistance to conventional drugs continues to increase. The proteasome represents a highly promising, actionable target to combat infectious diseases that devastate lives and livelihoods around the globe.

Deciphering mechanisms of immune escape to inform immunotherapeutic strategies in multiple myeloma.

Multiple myeloma is an incurable cancer characterized by the uncontrolled growth of malignant plasma cells nurtured within a permissive bone marrow microenvironment. While patients mount numerous adaptive immune responses directed against their disease, emerging data demonstrate that tumor intrinsic and extrinsic mechanisms allow myeloma cells to subvert host immunosurveillance and resist current therapeutic strategies. Myeloma downregulates antigens recognized by cellular immunity and modulates the bone marrow microenvironment to promote uncontrolled tumor proliferation, apoptotic resistance, and further hamper anti-tumor immunity. Additional resistance often develops after an initial clinical response to small molecules, immune-targeting antibodies, immune checkpoint blockade or cellular immunotherapy. Profound quantitative and qualitative dysfunction of numerous immune effector cell types that confer anti-myeloma immunity further supports myelomagenesis, disease progression and the emergence of drug resistance. Identification of tumor intrinsic and extrinsic resistance mechanisms may direct the design of rationally-designed drug combinations that prevent or overcome drug resistance to improve patient survival. Here, we summarize various mechanisms of immune escape as a means to inform novel strategies that may restore and improve host anti-myeloma immunity.

Novel therapies emerging in oncology to target the TGF-ß pathway.

The TGF-ß signaling pathway governs key cellular processes under physiologic conditions and is deregulated in many pathologies, including cancer. TGF-ß is a multifunctional cytokine that acts in a cell- and context-dependent manner as a tumor promoter or tumor suppressor. As a tumor promoter, the TGF-ß pathway enhances cell proliferation, migratory invasion, metastatic spread within the tumor microenvironment and suppresses immunosurveillance. Collectively, the pleiotropic nature of TGF-ß signaling contributes to drug resistance, tumor escape and undermines clinical response to therapy. Based upon a wealth of preclinical studies, the TGF-ß pathway has been pharmacologically targeted using small molecule inhibitors, TGF-ß-directed chimeric monoclonal antibodies, ligand traps, antisense oligonucleotides and vaccines that have been now evaluated in clinical trials. Here, we have assessed the safety and efficacy of TGF-ß pathway antagonists from multiple drug classes that have been evaluated in completed and ongoing trials. We highlight Vactosertib, a highly potent small molecule TGF-ß type 1 receptor kinase inhibitor that is well-tolerated with an acceptable safety profile that has shown efficacy against multiple types of cancer. The TGF-ß ligand traps Bintrafusp alfa (a bifunctional conjugate that binds TGF-ß and PD-L1), AVID200 (a computationally designed trap of TGF-ß receptor ectodomains fused to an Fc domain) and Luspatercept (a recombinant fusion that links the activin receptor IIb to IgG) offer new ways to fight difficult-to-treat cancers. While TGF-ß pathway antagonists are rapidly emerging as highly promising, safe and effective anticancer agents, significant challenges remain. Minimizing the unintentional inhibition of tumor-suppressing activity and inflammatory effects with the desired restraint on tumor-promoting activities has impeded the clinical development of TGF-ß pathway antagonists. A better understanding of the mechanistic details of the TGF-ß pathway should lead to more effective TGF-ß antagonists and uncover biomarkers that better stratify patient selection, improve patient responses and further the clinical development of TGF-ß antagonists.

Phase I Trial of Lithium and Tretinoin for Treatment of Relapsed and Refractory Non-promyelocytic Acute Myeloid Leukemia.

Glycogen synthase kinase-3 (GSK3) inhibitors induce differentiation and growth inhibition of acute myeloid leukemia (AML) cells. Our pre-clinical studies showed GSK3 inhibition leads to sensitization of AML cells to tretinoin-mediated differentiation. We conducted a phase I trial of lithium, a GSK3 inhibitor, plus tretinoin for relapsed, refractory non-promyelocytic AML. Nine patients with median (range) age 65 (42-82) years were enrolled. All subjects had relapsed leukemia after prior therapy, with a median (range) of 3 (1-3) prior therapies. Oral lithium carbonate 300 mg was given 2-3 times daily and adjusted to meet target serum concentration (0.6 to 1.0 mmol/L); tretinoin 22.5 or 45 mg/m2/day (two equally divided doses) was administered orally on days 1-7 and 15-21 of a 28-day cycle. Four patients attained disease stability with no increase in circulating blasts for ≥4 weeks. Median (range) survival was 106 days (60-502). Target serum lithium concentration was achieved in all patients and correlated with GSK3 inhibition in leukemic cells. Immunophenotypic changes associated with myeloid differentiation were observed in five patients. The combination treatment led to a reduction in the CD34+ CD38- AML stem cell population both in vivo and in vitro. The combination of lithium and tretinoin is well-tolerated, induces differentiation of leukemic cells, and may target AML stem cells, but has limited clinical activity in the absence of other antileukemic agents. The results of this clinical trial suggest GSK3 inhibition can result in AML cell differentiation and may be a novel therapeutic strategy in this disease, particularly in combination with other antileukemic agents. Lithium is a weak GSK3 inhibitor and future strategies in AML treatment will probably require more potent agents targeting this pathway or combinations with other antileukemic agents. This trial is registered at ClinicalTrials.gov NCT01820624.

Aberrant GSK3ß nuclear localization promotes AML growth and drug resistance.

Acute myeloid leukemia (AML) is a devastating disease with poor patient survival. As targetable mutations in AML are rare, novel oncogenic mechanisms are needed to define new therapeutic targets. We identified AML cells that exhibit an aberrant pool of nuclear glycogen synthase kinase 3ß (GSK3ß). This nuclear fraction drives AML growth and drug resistance. Nuclear, but not cytoplasmic, GSK3ß enhances AML colony formation and AML growth in mouse models. Nuclear GSK3ß drives AML partially by promoting nuclear localization of the NF-κB subunit, p65. Finally, nuclear GSK3ß localization has clinical significance as it strongly correlates to worse patient survival (n = 86; hazard ratio = 2.2; P < .01) and mediates drug resistance in cell and animal models. Nuclear localization of GSK3ß may define a novel oncogenic mechanism in AML and represent a new therapeutic target.

A Novel Glycogen Synthase Kinase-3 Inhibitor Optimized for Acute Myeloid Leukemia Differentiation Activity.

Standard therapies used for the treatment of acute myeloid leukemia (AML) are cytotoxic agents that target rapidly proliferating cells. Unfortunately, this therapeutic approach has limited efficacy and significant toxicity and the majority of AML patients still die of their disease. In contrast to the poor prognosis of most AML patients, most individuals with a rare subtype of AML, acute promyelocytic leukemia, can be cured by differentiation therapy using regimens containing all-trans retinoic acid. GSK3 has been previously identified as a therapeutic target in AML where its inhibition can lead to the differentiation and growth arrest of leukemic cells. Unfortunately, existing GSK3 inhibitors lead to suboptimal differentiation activity making them less useful as clinical AML differentiation agents. Here, we describe the discovery of a novel GSK3 inhibitor, GS87. GS87 was discovered in efforts to optimize GSK3 inhibition for AML differentiation activity. Despite GS87's dramatic ability to induce AML differentiation, kinase profiling reveals its high specificity in targeting GSK3 as compared with other kinases. GS87 demonstrates high efficacy in a mouse AML model system and unlike current AML therapeutics, exhibits little effect on normal bone marrow cells. GS87 induces potent differentiation by more effectively activating GSK3-dependent signaling components including MAPK signaling as compared with other GSK3 inhibitors. GS87 is a novel GSK3 inhibitor with therapeutic potential as a differentiation agent for non-promyelocytic AML. Mol Cancer Ther; 15(7); 1485-94. ©2016 AACR.

GSK-3 Inhibition Sensitizes Acute Myeloid Leukemia Cells to 1,25D-Mediated Differentiation.

1,25-dihydroxyvitamin D3 (1,25D), the biologically active form of vitamin D, is widely considered a promising therapy for acute myeloid leukemia (AML) based on its ability to drive differentiation of leukemic cells. However, clinical trials have been disappointing in part to dose-limiting hypercalcemia. Here we show how inhibiting glycogen synthase kinase 3 (GSK3) can improve the differentiation response of AML cells to 1,25D-mediated differentiation. GSK3 inhibition in AML cells enhanced the differentiating effects of low concentrations of 1,25D. In addition, GSK3 inhibition augmented the ability of 1,25D to induce irreversible growth inhibition and slow the progression of AML in mouse models. Mechanistic studies revealed that GSK3 inhibition led to the hyperphosphorylation of the vitamin D receptor (VDR), enabling an interaction between VDR and the coactivator, SRC-3 (NCOA3), thereby increasing transcriptional activity. We also found that activation of JNK-mediated pathways in response to GSK3 inhibition contributed to the potentiation of 1,25D-induced differentiation. Taken together, our findings offer a preclinical rationale to explore the repositioning of GSK3 inhibitors to enhance differentiation-based therapy for AML treatment. Cancer Res; 76(9); 2743-53. ©2016 AACR.

The role of TLR8 signaling in acute myeloid leukemia differentiation.

Acute myeloid leukemia (AML) is an aggressive disease with a poor 5-year survival of 21% that is characterized by the differentiation arrest of immature myeloid cells. For a rare subtype of AML (acute promyeloctyic leukemia, 5-10% of cases), all-trans retinoic acid therapy removes the differentiation block, yielding over a 90% cure rate. However, this treatment is not effective for the other 90-95% of AML patients, suggesting that new differentiation strategies are needed. Interestingly, differentiation is induced in normal hematopoietic cells through Toll-like receptor (TLR) stimulation and TLRs are expressed on AML cells. We present evidence that the TLR8 activation promotes AML differentiation and growth inhibition in a TLR8/MyD88/p38-dependent manner. We also show that that TLR7/TLR8 agonist, R848, considerably impairs the growth of human AML cells in immunodeficient mice. Our data suggests TLR8 activation has direct anti-leukemic effects independent of its immunomodulating properties that are currently under investigation for cancer therapy. Taken together, our results suggest that treatment with TLR8 agonists may be a promising new therapeutic strategy for AML.

Silencing near tRNA genes is nucleosome-mediated and distinct from boundary element function.

Transfer RNA (tRNA) genes and other RNA polymerase III transcription units are dispersed in high copy throughout nuclear genomes, and can antagonize RNA polymerase II transcription in their immediate chromosomal locus. Previous work in Saccharomyces cerevisiae found that this local silencing required subnuclear clustering of the tRNA genes near the nucleolus. Here we show that the silencing also requires nucleosome participation, though the nature of the nucleosome interaction appears distinct from other forms of transcriptional silencing. Analysis of an extensive library of histone amino acid substitutions finds a large number of residues that affect the silencing, both in the histone N-terminal tails and on the nucleosome disk surface. The residues on the disk surfaces involved are largely distinct from those affecting other regulatory phenomena. Consistent with the large number of histone residues affecting tgm silencing, survey of chromatin modification mutations shows that several enzymes known to affect nucleosome modification and positioning are also required. The enzymes include an Rpd3 deacetylase complex, Hos1 deacetylase, Glc7 phosphatase, and the RSC nucleosome remodeling activity, but not multiple other activities required for other silencing forms or boundary element function at tRNA gene loci. Models for communication between the tRNA gene transcription complexes and local chromatin are discussed.

Circulating CD4(+) and CD8(+) T cells are activated in inflammatory bowel disease and are associated with plasma markers of inflammation.

Inflammatory bowel disease (IBD) is characterized by damage to the gut mucosa and systemic inflammation. We sought to evaluate the role of chronic inflammation on circulating T-cell activation in human subjects with Crohn's disease and ulcerative colitis. We studied 54 patients with IBD and 28 healthy controls. T-cell activation and cycling were assessed in whole blood samples by flow cytometry. Levels of lipopolysaccharide (LPS) were measured in serum by Limulus amoebocyte lysate assay, and plasma levels of inflammatory markers and LPS-binding proteins were measured by ELISA. The proportions of circulating CD4(+) and CD8(+) T lymphocytes in cycle (Ki67(+) ) are increased in patients with IBD compared with these proportions in controls. CD8(+) T cells from patients with IBD are also enriched for cells that expressed CD38 and HLA-DR, and proportions of these cells are related to plasma levels of interleukin-6 and C-reactive protein in these patients. Intracellular interleukin-2 and interferon-γ levels were elevated in resting and polyclonally activated CD4(+) and CD8(+) T cells in patients with IBD when compared with levels from healthy controls. Surprisingly, we did not find increased levels of LPS in the serum of patients with IBD. We did, however, find a signature of recent microbial translocation, as levels of LPS-binding protein are increased in the plasma of patients with IBD compared with plasma levels in healthy controls; LPS-binding protein levels are also directly related to proportions of CD38 HLA-DR-expressing CD4(+) and CD8(+) T cells. Local damage to the gastrointestinal tract in IBD may result in systemic inflammation and T-cell activation.

Interleukin-12 is the optimum cytokine to expand human Th17 cells in vitro.

Recently, a new lineage of CD4+ T cells in humans and in mice has been reported. This T helper cell secretes interleukin-17 (IL-17) and has been defined as T helper 17 (Th17). Th17 cells express the IL-23 receptor (IL-23R) and play an important pathogenic role in different inflammatory conditions. In this study, our aim was to characterize the optimum conditions for isolation and propagation of human peripheral blood Th17 cells in vitro and the optimum conditions for isolation of Th17 clones. To isolate Th17 cells, two steps were taken. Initially, we negatively isolated CD4+ T cells from peripheral blood mononuclear cells of a normal human blood donor. Then, we isolated the IL-23R+ cells from the CD4+ T cells. Functional studies revealed that CD4+ IL-23R+ cells could be stimulated ex vivo with anti-CD3/CD28 to secrete both IL-17 and gamma interferon (IFN-gamma). Furthermore, we expanded the CD4+ IL-23R+ cells for 1 week in the presence of anti-CD3/CD28, irradiated autologous feeder cells, and different cytokines. Our data indicate that cytokine treatment increased the number of propagated cells 14- to 99-fold. Functional evaluation of the expanded number of CD4+ IL-23R+ cells in the presence of different cytokines with anti-CD3/CD28 revealed that all cytokines used (IL-2, IL-7, IL-12, IL-15, and IL-23) increased the amount of IFN-gamma secreted by IL-23R+ CD4+ cells at different levels. Our results indicate that IL-7 plus IL-12 was the optimum combination of cytokines for the expansion of IL-23R+ CD4+ cells and the secretion of IFN-gamma, while IL-12 preferentially stimulated these cells to secrete predominately IL-17.