Cancer immunotherapy has shown promising clinical outcomes in many patients. However, some patients still fail to respond, and new strategies are needed to overcome resistance. The purpose of this ...study was to identify novel genes and understand the mechanisms that confer resistance to cancer immunotherapy.
To identify genes mediating resistance to T-cell killing, we performed an open reading frame (ORF) screen of a kinome library to study whether overexpression of a gene in patient-derived melanoma cells could inhibit their susceptibility to killing by autologous tumor-infiltrating lymphocytes (TIL).
The RNA-binding protein MEX3B was identified as a top candidate that decreased the susceptibility of melanoma cells to killing by TILs. Further analyses of anti-PD-1-treated melanoma patient tumor samples suggested that higher
expression is associated with resistance to PD-1 blockade. In addition, significantly decreased levels of IFNγ were secreted from TILs incubated with MEX3B-overexpressing tumor cells. Interestingly, this phenotype was rescued upon overexpression of exogenous HLA-A2. Consistent with this, we observed decreased HLA-A expression in MEX3B-overexpressing tumor cells. Finally, luciferase reporter assays and RNA-binding protein immunoprecipitation assays suggest that this is due to MEX3B binding to the 3' untranslated region (UTR) of
to destabilize the mRNA.
MEX3B mediates resistance to cancer immunotherapy by binding to the 3' UTR of
to destabilize the
mRNA and thus downregulate HLA-A expression on the surface of tumor cells, thereby making the tumor cells unable to be recognized and killed by T cells.
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Proteasome inhibitors have revolutionized outcomes in multiple myeloma, but they are used empirically, and primary and secondary resistance are emerging problems. We have identified TJP1 as a ...determinant of plasma cell proteasome inhibitor susceptibility. TJP1 suppressed expression of the catalytically active immunoproteasome subunits LMP7 and LMP2, decreased proteasome activity, and enhanced proteasome inhibitor sensitivity in vitro and in vivo. This occurred through TJP1-mediated suppression of EGFR/JAK1/STAT3 signaling, which modulated LMP7 and LMP2 levels. In the clinic, high TJP1 expression in patient myeloma cells was associated with a significantly higher likelihood of responding to bortezomib and a longer response duration, supporting the use of TJP1 as a biomarker to identify patients most likely to benefit from proteasome inhibitors.
•TJP1 expression is a biomarker of proteasome inhibitor sensitivity in myeloma•Signaling through the TJP1/EGFR/JAK/STAT pathway influences proteasome capacity•EGFR/JAK/STAT suppression induces chemosensitization to proteasome inhibitors
Zhang et al. show that TJP1 enhances multiple myeloma sensitivity to proteasome inhibitors by reducing the expression of the immunoproteasome subunits LMP7 and LMP2 via suppression of EGFR/JAK1/STAT3 signaling, and that high TJP1 expression in patient myeloma cells correlates with better response to bortezomib.
Mesenchymal stromal cells (MSCs) are a major component of the leukemia bone marrow (BM) microenvironment. Connective tissue growth factor (CTGF) is highly expressed in MSCs, but its role in the BM ...stroma is unknown. Therefore, we knocked down (KD) CTGF expression in human BM-derived MSCs by CTGF short hairpin RNA. CTGF KD MSCs exhibited fivefold lower proliferation compared with control MSCs and had markedly fewer S-phase cells. CTGF KD MSCs differentiated into adipocytes at a sixfold higher rate than controls in vitro and in vivo. To study the effect of CTGF on engraftment of leukemia cells into BM, an in vivo model of humanized extramedullary BM (EXM-BM) was developed in NOD/SCID/IL-2rgnull mice. Transplanted Nalm-6 or Molm-13 human leukemia cells engrafted at a threefold higher rate in adipocyte-rich CTGF KD MSC-derived EXM-BM than in control EXM-BM. Leptin was found to be highly expressed in CTGF KD EXM-BM and in BM samples of patients with acute myeloid and acute lymphoblastic leukemia, whereas it was not expressed in normal controls. Given the established role of the leptin receptor in leukemia cells, the data suggest an important role of CTGF in MSC differentiation into adipocytes and of leptin in homing and progression of leukemia.
•Connective tissue growth factor regulates adipogenic differentiation of MSCs.•Connective tissue growth factor regulates leukemia engraftment.
Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the aberrant expression of several growth-regulating, oncogenic effectors. Exportin 1 (XPO1) mediates the ...nucleocytoplasmic transport of numerous molecules including oncogenic growth-regulating factors, RNAs, and ribosomal subunits. In MCL cells, the small molecule KPT-185 blocks XPO1 function and exerts anti-proliferative effects. In this study, we investigated the molecular mechanisms of this putative anti-tumor effect on MCL cells using cell growth/viability assays, immunoblotting, gene expression analysis, and absolute quantification proteomics. KPT-185 exhibited a p53-independent anti-lymphoma effect on MCL cells, by suppression of oncogenic mediators (e.g., XPO1, cyclin D1, c-Myc, PIM1, and Bcl-2 family members), repression of ribosomal biogenesis, and downregulation of translation/chaperone proteins (e.g., PIM2, EEF1A1, EEF2, and HSP70) that are part of the translational/transcriptional network regulated by heat shock factor 1. These results elucidate a novel mechanism in which ribosomal biogenesis appears to be a key component through which XPO1 contributes to tumor cell survival. Thus, we propose that the blockade of XPO1 could be a promising, novel strategy for the treatment of MCL and other malignancies overexpressing XPO1.
BackgroundChimeric antigen receptor (CAR) T cells targeting CD19 mediate potent and durable effects in B-cell malignancies. However, antigen loss or downregulation is a frequent cause of resistance. ...Here, we report development of a novel CAR T-cell therapy product to target CD79b, a pan B-cell antigen, widely expressed in most B-cell lymphomas.MethodsWe generated a novel anti-CD79b monoclonal antibody by hybridoma method. The specificity of the antibody was determined by testing against isogenic cell lines with human CD79b knock-in or knock-out. A single-chain variable fragment derived from the monoclonal antibody was used to make a panel of CD79b-targeting CAR molecules containing various hinge, transmembrane, and co-stimulatory domains. These were lentivirally transduced into primary T cells and tested for antitumor activity in in vitro and in vivo B-cell lymphoma models.ResultsWe found that the novel anti-CD79b monoclonal antibody was highly specific and bound only to human CD79b and no other cell surface protein. In testing the various CD79b-targeting CAR molecules, superior antitumor efficacy in vitro and in vivo was found for a CAR consisting CD8α hinge and transmembrane domains, an OX40 co-stimulatory domain, and a CD3ζ signaling domain. This CD79b CAR specifically recognized human CD79b-expressing lymphoma cell lines but not CD79b knock-out cell lines. CD79b CAR T cells, generated from T cells from either healthy donors or patients with lymphoma, proliferated, produced cytokines, degranulated, and exhibited robust cytotoxic activity in vitro against CD19+ and CD19– lymphoma cell lines and patient-derived lymphoma tumors relapsing after prior CD19 CAR T-cell therapy. Furthermore, CD79b CAR T cells were highly efficient at eradicating pre-established lymphoma tumors in vivo in three aggressive lymphoma xenograft models, including two cell line-derived xenografts and one patient-derived xenograft. Notably, these CAR T cells did not demonstrate any significant tonic signaling activity or markers of exhaustion.ConclusionOur results indicated that this novel CD79b CAR T-cell therapy product has robust antitumor activity against B-cell lymphomas. These results supported initiation of a phase 1 clinical trial to evaluate this product in patients with relapsed or refractory B-cell lymphomas.
Abstract
Loss of IL-15 expression by colon tumors strongly correlates with classification as microsatellite stable, decreased tumor infiltrating lymphocytes (TILs), increased metastasis, and poor ...responses to immunotherapy. To better understand how IL-15 expression regulates immune cells in the tumor microenvironment (TME) and identify the importance of IL-15 expressed by tumor cells, we developed a mouse model system to examine this by generating MC-38 tumor cells deficient in IL-15 using CRISPR/Cas9 system. After one round of IL-15 deletion, MC-38 cells that lack one IL-15 allele (i.e IL-15+/− MC-38 cells) were identified and expressed decreased levels of soluble IL-15 complexes. Surprisingly, deletion of one IL-15 allele was sufficient to affect tumor growth as IL-15+/− MC-38 tumors grew faster than Wt MC-38 tumors. IL-15+/− MC-38 tumors had decreased CD8 and NK TILs and increased CD11b+Ly6G+ and CD11b+Ly6ChiLy6G− myeloid cells than Wt tumors. Gene expression analysis of myeloid cells showed that blocking IL-15 in tumors increased expression of CD206, ARG1, and TDO2 and decreased IL-12β and IL-6. A second round of deletion led to complete deletion of IL-15 in MC-38 cells (i.e. IL-15−/− MC-38) and upon implantation developed tumors with further decreases in CD8 and NK TILs. In mice lacking IL-15Rα in the intestinal epithelium, AOM/DSS-induced colon tumors also harbored decreased numbers of CD8 TILs. These results demonstrate that even partially decreasing levels of IL-15 in the TME negatively impacts the numbers of CD8 and NK TILs and skews myeloid cells towards a pro-tumor landscape. Overall, these models that can be used to identify the mechanisms contributing to the poor therapeutic responses observed in human colon carcinomas.
Metabolic reprogramming has been described as a hallmark of transformed cancer cells. In this study, we examined the role of the glutamine (Gln) utilization pathway in acute myeloid leukemia (AML) ...cell lines and primary AML samples. Our results indicate that a subset of AML cell lines is sensitive to Gln deprivation. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes the conversion of Gln to glutamate. One of the two GLS isoenzymes, GLS1 is highly expressed in cancer and encodes two different isoforms: kidney (KGA) and glutaminase C (GAC). We analyzed mRNA expression of GLS1 splicing variants, GAC and KGA, in several large AML datasets and identified increased levels of expression in AML patients with complex cytogenetics and within specific molecular subsets. Inhibition of glutaminase by allosteric GLS inhibitor bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide or by novel, potent, orally bioavailable GLS inhibitor CB-839 reduced intracellular glutamate levels and inhibited growth of AML cells. In cell lines and patient samples harboring IDH1/IDH2 (Isocitrate dehydrogenase 1 and 2) mutations, CB-839 reduced production of oncometabolite 2-hydroxyglutarate, inducing differentiation. These findings indicate potential utility of glutaminase inhibitors in AML therapy, which can inhibit cell growth, induce apoptosis and/or differentiation in specific leukemia subtypes.
CXCR5 mediates homing of both B and follicular helper T (T
) cells into follicles of secondary lymphoid organs. We found that CXCR5
CD8
T cells are present in human tonsils and follicular lymphoma, ...inhibit T
-mediated B cell differentiation, and exhibit strong cytotoxic activity. Consistent with these findings, adoptive transfer of CXCR5
CD8
T cells into an animal model of lymphoma resulted in significantly greater antitumor activity than CXCR5
CD8
T cells. Furthermore, RNA-Seq-based transcriptional profiling revealed 77 differentially expressed genes unique to CXCR5
CD8
T cells. Among these, a signature comprised of 33 upregulated genes correlated with improved survival in follicular lymphoma patients. We also showed that CXCR5
CD8
T cells could be induced and expanded ex vivo using IL-23 plus TGF-β, suggesting a possible strategy to generate these cells for clinical application. In summary, our study identified CXCR5
CD8
T cells as a distinct T cell subset with ability to suppress T
-mediated B cell differentiation, exert strong antitumor activity, and confer favorable prognosis in follicular lymphoma patients.
Cutaneous squamous cell carcinoma (cSCC) comprises 15‒20% of all skin cancers and has a well-defined progression sequence from precancerous actinic keratosis to invasive cSCC. To identify targets for ...chemoprevention, we previously reported a cross-species analysis to identify the transcriptional drivers of cSCC development and identified miR-181a as a potential oncomiR. We show that the upregulation of miR-181a promotes multiple protumorigenic properties by targeting an understudied component of TGFβ signaling, TGFβR3. miR-181a and TGFβR3 are upregulated and downregulated, respectively, in cSCC. miR-181a overexpression (OE) and TGFβR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT cells and in primary normal human epidermal keratinocytes. In addition, OE of miR-181a or KD of TGFβR3 by short hairpin RNA enhances anchorage-independent survival. miR-181a OE or TGFβR3 KD enhances cellular migration and invasion and upregulation of epithelial‒mesenchymal transition markers. Luciferase reporter assays demonstrate that miR-181a directly targets the 3′-untranslated region of TGFβR3. miR-181a upregulates phosphorylated SMAD3 levels after TGFβ2 administration and results in elevated SNAIL and SLUG expression. Finally, we confirm in vivo that miR-181a inhibition compromises tumor growth. Importantly, these phenotypes can be reversed with TGFβR3 OE or KD in the context of miR-181a OE or KD, respectively, further highlighting the physiologic relevance of this regulation in cSCC.
Summary
This phase 2 study evaluated the activity and safety of ibrutinib, a Bruton’s tyrosine kinase inhibitor, plus rituximab in adults with previously untreated follicular lymphoma. Patients ...received once‐daily ibrutinib 560 mg continuously plus once‐weekly rituximab 375 mg/m2 for 4 weeks beginning Week 1 (Arm 1, n = 60) or Week 9 (following an 8‐week ibrutinib lead‐in) to explore biomarkers (Arm 2, n = 20). The primary endpoint was the best overall response rate (ORR). The median age was 58 years; most had an Eastern Cooperative Oncology Group Performance Status of 0 (74%) and Stage III/IV disease (84%). At a median study follow‐up of 34 months in Arm 1 and 29 months in Arm 2, ORRs were 85% 95% confidence interval (CI) 73–93 and 75% (95% CI 51–91), respectively, with complete responses in 40% and 50%. The median duration of response was not reached in either arm; 30‐month progression‐free and overall survival rates were 67% and 97% (Arm 1) and 65% and 100% (Arm 2). The most common adverse events were fatigue, diarrhoea and nausea. Higher grade (Grade 3/4) haematological, haemorrhagic and cardiac events occurred infrequently. Ibrutinib plus rituximab was active and tolerable in first‐line follicular lymphoma.
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