SHP2 inhibitors offer an appealing and novel approach to inhibit receptor tyrosine kinase (RTK) signaling, which is the oncogenic driver in many tumors or is frequently feedback activated in response ...to targeted therapies including RTK inhibitors and MAPK inhibitors. We seek to evaluate the efficacy and synergistic mechanisms of combinations with a novel SHP2 inhibitor, TNO155, to inform their clinical development.
The combinations of TNO155 with EGFR inhibitors (EGFRi), BRAFi, KRAS
i, CDK4/6i, and anti-programmed cell death-1 (PD-1) antibody were tested in appropriate cancer models
and
, and their effects on downstream signaling were examined.
In EGFR-mutant lung cancer models, combination benefit of TNO155 and the EGFRi nazartinib was observed, coincident with sustained ERK inhibition. In BRAF
colorectal cancer models, TNO155 synergized with BRAF plus MEK inhibitors by blocking ERK feedback activation by different RTKs. In KRAS
cancer cells, TNO155 effectively blocked the feedback activation of wild-type KRAS or other RAS isoforms induced by KRAS
i and greatly enhanced efficacy. In addition, TNO155 and the CDK4/6 inhibitor ribociclib showed combination benefit in a large panel of lung and colorectal cancer patient-derived xenografts, including those with KRAS mutations. Finally, TNO155 effectively inhibited RAS activation by colony-stimulating factor 1 receptor, which is critical for the maturation of immunosuppressive tumor-associated macrophages, and showed combination activity with anti-PD-1 antibody.
Our findings suggest TNO155 is an effective agent for blocking both tumor-promoting and immune-suppressive RTK signaling in RTK- and MAPK-driven cancers and their tumor microenvironment. Our data provide the rationale for evaluating these combinations clinically.
CD4⁺ T helper 1 ($\text{T}_{\text{H}}1$) cells are important mediators of inflammation and are regulated by numerous pathways, including the negative immune receptor Tim-3. We found that Tim-3 is ...constitutively expressed on cells of the innate immune system in both mice and humans, and that it can synergize with Toll-like receptors. Moreover, an antibody agonist of Tim-3 acted as an adjuvant during induced immune responses, and Tim-3 ligation induced distinct signaling events in T cells and dendritic cells; the latter finding could explain the apparent divergent functions of Tim-3 in these cell types. Thus, by virtue of differential expression on innate versus adaptive immune cells, Tim-3 can either promote or terminate$\text{T}_{\text{H}}1$immunity and may be able to influence a range of inflammatory conditions.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The recent discovery of CD4(+) T cells characterized by secretion of interleukin (IL)-17 (T(H)17 cells) and the naturally occurring regulatory FOXP3(+) CD4 T cell (nT(reg)) has had a major impact on ...our understanding of immune processes not readily explained by the T(H)1/T(H)2 paradigm. T(H)17 and nT(reg) cells have been implicated in the pathogenesis of human autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and psoriasis. Our recent data and the work of others demonstrated that transforming growth factor-beta (TGF-beta) and IL-6 are responsible for the differentiation of naive mouse T cells into T(H)17 cells, and it has been proposed that IL-23 may have a critical role in stabilization of the T(H)17 phenotype. A second pathway has been discovered in which a combination of TGF-beta and IL-21 is capable of inducing differentiation of mouse T(H)17 cells in the absence of IL-6 (refs 6-8). However, TGF-beta and IL-6 are not capable of differentiating human T(H)17 cells and it has been suggested that TGF-beta may in fact suppress the generation of human T(H)17 cells. Instead, it has been recently shown that the cytokines IL-1beta, IL-6 and IL-23 are capable of driving IL-17 secretion in short-term CD4(+) T cell lines isolated from human peripheral blood, although the factors required for differentiation of naive human CD4 to T(H)17 cells are still unknown. Here we confirm that whereas IL-1beta and IL-6 induce IL-17A secretion from human central memory CD4(+) T cells, TGF-beta and IL-21 uniquely promote the differentiation of human naive CD4(+) T cells into T(H)17 cells accompanied by expression of the transcription factor RORC2. These data will allow the investigation of this new population of T(H)17 cells in human inflammatory disease.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
SHP2 is a ubiquitous tyrosine phosphatase involved in regulating both tumor and immune cell signaling. In this study, we discovered a novel immune modulatory function of SHP2. Targeting this protein ...with allosteric SHP2 inhibitors promoted anti-tumor immunity, including enhancing T cell cytotoxic function and immune-mediated tumor regression. Knockout of SHP2 using CRISPR/Cas9 gene editing showed that targeting SHP2 in cancer cells contributes to this immune response. Inhibition of SHP2 activity augmented tumor intrinsic IFNγ signaling resulting in enhanced chemoattractant cytokine release and cytotoxic T cell recruitment, as well as increased expression of MHC Class I and PD-L1 on the cancer cell surface. Furthermore, SHP2 inhibition diminished the differentiation and inhibitory function of immune suppressive myeloid cells in the tumor microenvironment. SHP2 inhibition enhanced responses to anti-PD-1 blockade in syngeneic mouse models. Overall, our study reveals novel functions of SHP2 in tumor immunity and proposes that targeting SHP2 is a promising strategy for cancer immunotherapy.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
TIM‐3 is a molecule selectively expressed on a subset of murine IFN‐γ‐secreting T helper 1 (Th1) cells but not Th2 cells, and regulates Th1 immunity and tolerance in vivo. At this time little is ...known about the role of TIM‐3 on human T cells. To determine if TIM‐3 similarly identifies and regulates Th1 cells in humans, we generated a panel of mAb specific for human TIM‐3. We report that TIM‐3 is expressed by a subset of activated CD4+ cells, and that anti‐CD3/anti‐CD28 stimulation increases both the level of expression as well as the number of TIM‐3+ T cells. We also find that TIM‐3 is expressed at high levels on in vitro polarized Th1 cells, and is expressed at lower levels on Th17 cells. In addition, human CD4+ T cells secreted elevated levels of IFN‐γ, IL‐17, IL‐2, and IL‐6, but not IL‐10, IL‐4, or TNF‐α, when stimulated with anti‐CD3/anti‐CD28 in the presence of TIM‐3‐specific, putative antagonistic antibodies. This was not mediated by differences in proliferation or cell death, but rather by induction of cytokines at the transcriptional level. These results suggest that TIM‐3 is a negative regulator of human T cells and regulates Th1 and Th17 cytokine secretion.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed ...cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.
The secretion of IL-9, initially recognized as a Th2 cytokine, was recently attributed to a novel CD4 T cell subset termed Th9 in the murine system. However, IL-9 can also be secreted by mouse Th17 ...cells and may mediate aspects of the proinflammatory activities of Th17 cells. Here we report that IL-9 is secreted by human naive CD4 T cells in response to differentiation by Th9 (TGF-beta and IL-4) or Th17 polarizing conditions. Yet, these differentiated naive cells did not coexpress IL-17 and IL-9, unless they were repeatedly stimulated under Th17 differentiation-inducing conditions. In contrast to the naive cells, memory CD4 T cells were induced to secrete IL-9 by simply providing TGF-beta during stimulation, as neither IL-4 nor proinflammatory cytokines were required. Furthermore, the addition of TGF-beta to the Th17-inducing cytokines (IL-1beta, IL-6, IL-21, IL-23) that induce memory cells to secrete IL-17, resulted in the marked coexpression of IL-9 in IL-17 producing memory cells. The proinflammatory cytokine mediating TGF-beta-dependent coexpression of IL-9 and IL-17 was identified to be IL-1beta. Moreover, circulating monocytes were potent costimulators of IL-9 production by Th17 cells via their capacity to secrete IL-1beta. Finally, to determine whether IL-9/IL-17 coproducing CD4 cells were altered in an inflammatory condition, we examined patients with autoimmune diabetes and demonstrated that these subjects exhibit a higher frequency of memory CD4 cells with the capacity to transition into IL-9(+)IL-17(+) cells. These data demonstrate the presence of IL-17(+)IL-9(+) CD4 cells induced by IL-1beta that may play a role in human autoimmune disease.
B‐1 cells constitute a distinct B cell population with unique phenotypic and functional characteristics. Although the origin of B‐1 cells remains controversial, B‐1 cells in different locations are ...generally considered to be part of the same pool. To determine the validity of this assumption, we examined peritoneal and splenic B‐1 cells isolated by flow cytometric cell sorting from normal mice for several features. We found that splenic B‐1 cells differ from peritoneal B‐1 cells in terms of surface antigen expression, viability ex vivo, immunoglobulin secretion in vitro, stimulated cell cycle progression, and expression of Notch family, Notch‐dependent, and Notch‐associated genes. These results indicate that splenic and peritoneal B‐1 cells are not the same and thus dispute the notion that B‐1 cells are uniform, and may suggest that different subpopulations of B‐1 cells arise separately, home individually, and/or are heavily influenced by local environmental factors.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
B‐1 and B‐2 cells are lymphocyte populations that differ in development, surface marker expression, tissue localization, and function. Though mainly found in the spleen, lymph nodes, and circulation ...of mice, small numbers of B‐2 cells are found in the peritoneal cavity, a site predominantly populated by B‐1 cells. Here, we characterized peritoneal B‐2 cells, and determined their relationship to B‐1 cells. We found that peritoneal B‐2 cells appear to be intermediate between splenic B‐2 and peritoneal B‐1 cells in terms of surface marker expression of B220, CD80, and CD43, expression of several marker genes, and in vitro viability and IgM secretion. Adoptive transfer of peritoneal B‐2 cells into severe combined immunodeficiency mice resulted in the acquisition of a phenotype reminiscent of B‐1b cells, as shown by up‐regulation of Mac‐1 and CD43, and down‐regulation of CD23. Moreover, adoptively transferred peritoneal B‐2 cells recapitulated B‐1 cell function by producing natural IgM in recipient mice. These data suggest that peritoneal B‐2 cells express some characteristics of B‐1b cells and that this similarity increases with additional time in the peritoneal cavity.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
RAD51D is a key player in DNA repair by homologous recombination (HR), and
truncating variant carriers have an increased risk for ovarian cancer. However, the contribution of nontruncating
variants ...to cancer predisposition remains uncertain. Using deep sequencing and case-control genotyping studies, we show that in French Canadians, the missense
variant c.620C>T;p.S207L is highly prevalent and is associated with a significantly increased risk for ovarian high-grade serous carcinoma (HGSC; 3.8% cases vs. 0.2% controls). The frequency of the p.S207L variant did not significantly differ from that of controls in breast, endometrial, pancreas, or colorectal adenocarcinomas. Functionally, we show that this mutation impairs HR by disrupting the RAD51D-XRCC2 interaction and confers PARP inhibitor sensitivity. These results highlight the importance of a functional RAD51D-XRCC2 interaction to promote HR and prevent the development of HGSC. This study identifies c.620C>T;p.S207L as the first bona fide pathogenic
missense cancer susceptibility allele and supports the use of targeted PARP-inhibitor therapies in ovarian cancer patients carrying deleterious missense
variants.
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