Blockade Of TGF-β Signaling Research Articles

A phase II double window of opportunity trial evaluating the oral TGF-beta receptor I inhibitor vactosertib in patients undergoing standard of care preoperative therapy for locally advanced esophageal adenocarcinoma.

TPS516 Background: Esophageal adenocarcinoma (EAC) is the seventh most common cancer globally and ranks sixth in overall mortality. Despite improvements in pre-operative therapy, EAC has a high recurrence rate, especially among patients whose tumors do not achieve pathologic complete response (pCR), thus there is a critical need to enhance pCR rate and improve long-term outcomes. We recently discovered that a subset of EACs are driven by tumor-intrinsic oncogenic TGFβ signaling and have identified two potential biomarkers to predict response to anti-TGFβ therapy. One of these biomarkers is enriched in poorly differentiated EAC. We hypothesize that TGFβ signaling blockade will exert a tumor-inhibitory effect in patients with EAC and propose a double window-of-opportunity trial to test if TGFβ receptor I inhibitor vactosertib can improve pCR and induce tumor metabolic response in patients undergoing neoadjuvant therapy for localized EAC. Methods: This phase II double window of opportunity study assesses the efficacy and pharmacodynamics of the oral TGFβ Receptor I inhibitor vactosertib in patients with locally advanced esophageal adenocarcinoma (EAC). Patients will be treated with single-agent vactosertib in two windows of opportunity: the first will be prior to initiation of standard of care neoadjuvant therapy and the second after completion of neoadjuvant therapy prior to surgery. Patients will be eligible if they have poorly differentiated EAC and are appropriate for neoadjuvant therapy followed by resection as per standard of care. The primary objectives are improvement in pCR compared to historical controls and metabolic response (decrease in fluorodeoxyglucose uptake by PET CT) in the primary tumor after the first window. Secondary objectives will evaluate biomarkers of response identified in preclinical studies. Pre- and post-first window of opportunity treatment biopsies will be obtained for correlative and exploratory analyses. Clinical trial information: NCT06044311 .

Potentiating CAR-T cell function in the immunosuppressive tumor microenvironment by inverting the TGF-β signal.

The immunosuppressive tumor microenvironment represents a key challenge for chimeric antigen receptor (CAR) Tcells in solid tumors and includes the production of the inhibitory cytokine transforming growth factor β (TGF-β), which limits CAR-T cell persistence and function. Current strategies involving the blockade of TGF-β signaling have little benefit for solid tumor treatment. Here, we demonstrate a novel inverted cytokine receptor (ICR)-modified CAR-T cell strategy not only TGF-β signal blockade but also antitumor efficacy enhancement. The newly designed Tcells carry an ICR construct that fuses the TGF-β receptor II extracellular domain to the interleukin-15 (IL-15) receptor α cytoplasmic domain (named TB15) and is directed to the tumor antigen epidermal growth factor receptor by a CAR construct. In mice with high TGF-β solid tumors, our signal-inverted CAR/TB15 Tcells effectively treat tumors by blocking TGF-β and repurposing IL-15 stimulative signaling, resulting in enhanced CAR-T cell persistence and function. As a proof of concept, our study results extend synthetic receptor signaling beyond CAR-directed killing, which could endow adoptively transferred Tcells with new functions that overcome major barriers in the treatment of solid tumors by using a chimeric ICR.

P-1866. Polymorphonuclear Myeloid-Derived Suppressor Cells regulates immune reconstitution during HIV infection through PD-L1 and TGF-β signaling

Abstract Background Although myeloid-derived suppressor cells (MDSCs) are widely recognized for their immunoinhibitory effect in a variety of pathological conditions, their function during human immunodeficiency virus (HIV) infection and the onset of inadequate immune reconstitution remains elusive. PMN-MDSCs inhibit autologous CD4+ T-cell proliferation and cytokines (IL-2, TNF-α, and IFN-γ) production in HIV INRs. Methods We conducted a cross-sectional study in which 30 healthy controls (HC) and 62 HIV-1-infected subjects [31 immunological non-responders (INRs) and 31 immunological responders (IRs)] were selected. After two years of antiretroviral therapy, INRs and IRs were designated as patients with CD4+ T-cell counts < 350 cells/μL and >500 cells/μL, respectively. The proportion of MDSCs was measured and their relationship with HIV disease progression was studied. Specifically, using flow cytometry and real-time PCR, immune regulatory molecules (including programmed death-ligand 1 [PD-L1], arginase 1, inducible nitric oxide synthase, interleukin 10, transforming growth factor beta [TGF-β], and indoleamine 2,3-dioxygenase) that are relevant for MDSCs activity were quantified. Furthermore, we investigated the impact of the blockade of PD-L1 and/or TGF-β signaling on MDSCs and their effects on CD4+ T cells using in vitro functional experiments. PD-L1 and TGF-β mediate the suppressive effect of PMN-MDSCs on CD4+ T-cells response. Results We found that polymorphonuclear MDSCs (PMN-MDSCs) are more abundant and negatively correlated with CD4+ T-cell counts in HIV-infected individuals. PMN-MDSCs suppress CD4+ T-cell proliferation and interferon-γ (IFN-γ) production in INRs. Furthermore, correlations were found between PD-L1 expression on PMN-MDSCs and PD-1+ CD4+ T-cells. TGF-β expression on PMN-MDSCs was likewise enhanced in INRs. Importantly, inhibiting both PD-L1 and TGF-β signaling had a synergistic impact on restoring CD4+ T-cell activity in vitro. Conclusion Our findings reveal that the expansion of PMN-MDSCs decreased CD4+ T-cell proliferation and IFN-γ secretion in HIV infection through PD-L1 and TGF-β mediated pathways. Interventions that target both PD-L1 and TGF-β pathways may be novel strategies for enhancing immune reconstitution in INRs. Disclosures All Authors: No reported disclosures

Disruption of TGF-β signaling pathway is required to mediate effective killing of hepatocellular carcinoma by human iPSC-derived NK cells

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Transforming growth factor beta (TGF-β) is highly expressed in the liver tumor microenvironment and is known to inhibit immune cell activity. Here, we used human induced pluripotent stem cells (iPSCs) to produce natural killer (NK) cells engineered to mediate improved anti-HCC activity. Specifically, we produced iPSC-NK cells with either knockout TGF-β receptor 2 (TGFBR2-KO) or expression of a dominant negative (DN) form of the TGF-β receptor 2 (TGFBR2-DN) combined with chimeric antigen receptors (CARs) that target either GPC3 or AFP. The TGFBR2-KO and TGFBR2-DN iPSC-NK cells are resistant to TGF-β inhibition and improved anti-HCC activity. However, expression of anti-HCC CARs on iPSC-NK cells did not lead to effective anti-HCC activity unless there was also inhibition of TGF-β activity. Our findings demonstrate that TGF-β signaling blockade is required for effective NK cell function against HCC and potentially other malignancies that express high levels of TGF-β.

Adrenal gland macrophages regulate glucocorticoid production through Trem2 and TGF-β.

Glucocorticoid synthesis by adrenal glands (AGs) is regulated by the hypothalamic-pituitary-adrenal axis to facilitate stress responses when the host is exposed to stimuli. Recent studies implicate macrophages as potential steroidogenic regulators, but the molecular mechanisms by which AG macrophages exert such influence remain unclear. In this study, we investigated the role of AG macrophages in response to cold challenge or atherosclerotic inflammation as physiologic models of acute or chronic stress. Using single-cell RNA sequencing, we observed dynamic AG macrophage polarization toward classical activation and lipid-associated phenotypes following acute or chronic stimulation. Among transcriptional alterations induced in macrophages, triggering receptor expressed on myeloid cells 2 (Trem2) was highlighted because of its upregulation following stress. Conditional deletion of macrophage Trem2 revealed a protective role in stress responses. Mechanistically, Trem2 deletion led to increased AG macrophage death, abolished the TGF-β-producing capacity of AG macrophages, and resulted in enhanced glucocorticoid production. In addition, enhanced glucocorticoid production was replicated by blockade of TGF-β signaling. Together, these observations suggest that AG macrophages restrict steroidogenesis through Trem2 and TGF-β, which opens potential avenues for immunotherapeutic interventions to resolve stress-related disorders.

Abstract 3615: Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells

Abstract Background: Neuroblastoma (NB) is a common malignant extracranial solid tumor in children, responsible for 11% of cancer deaths in pediatric patients under 15 (Smith et al, Cancer, 2014). High-risk neuroblastoma is a metastatic disease in children aged 18 months or older with L2, M, or MS disease with amplification of the MYCN oncogene (Chuang Pediatr Blood Cancer 2021). Recent analyses by COG show that HR Neuroblastoma still has a 5-year event free survival of 51% despite an extensive treatment regimen (Steven et al, ASCO, 2022). To address improved tumor targeting and overcome antigen escape, chimeric antigen receptor (CAR) technology to Natural Killer (NK) cell therapy has been adapted (Chu/Cairo, et al, Frontiers in Oncology, 2019). TGFβ is a potent mechanism of the NK cell anti-tumor resistance and is present at elevated levels in the NB tumor microenvironment (TME) (Foltz, Cancers, 2018). Studies demonstrated that blockade of TGF-β signaling in NK cells caused the accumulation of NK cells that produce IFN-γ and neutralization of TGF-β prevented NKG2D downregulation and also restored NK cell anti-tumor reactivity (Nayyar/Chu/Cairo, et al Frontierls in Oncology, 2018). ROR1, a transmembrane protein is highly expressed on the majority of NB (Rebagay/Yan Front Oncol, 2012). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (Chu/Cairo, et al Cancer Immunol Res, 2015). Objective: To determine in vitro cytotoxicity against neuroblastoma cells by ex vivo expanded TGFβ imprinting (TGFβi) NK cells. Methods: TGFβi NK cells are generated by culturing expanded NK cells with irradiated K562-41BBL-mbIL21 in 50 IU/mL IL-2 plus 10 ng/mL TGFβ every 2-3 days for two weeks. NK and TGFβi NK cells specific in-vitro cytotoxicity were compared via luciferase reporter assay against ROR1+ NB cell lines as we previously described (Chu/Cairo, et al, JITC, 2022). Cytokine/chemokine levels secreted by NK and TGFβi NK cells were compared using ELISA assays (Chu/Cairo, et al, JITC, 2022). Results: We have successfully expanded TGFβi NK cells. These TGFβi NK cells showed significantly enhanced in vitro cytotoxicity against NB cells compared to NK cells under TGFβ environment (20+2.4% vs 0%; p<0.001) at E:T=3:1 and consistently, TGFβ-imprinting NK secreted significantly high level of TNF-alpha (393.7pg/ml+16.77pg/ml) against NB cells (p<0.001) compared to NK cells (79.7pg/ml+14.7pg/ml) under TGFβ environment. Conclusion: Our data indicate TGFβi NK cells may be a promising novel therapy to overcome the immunosuppression of TGFβ in the tumor microenvironment for pediatric NB tumors. We will further engineer these TGFβi NK cells to express anti-ROR1 CAR to enhance the targeting specificity against NB tumors and investigate the anti-tumor effects of anti-ROR1 CAR TGFβi NK cells against NB tumors in-vitro and in-vivo (Funded by U54 CA232561). Citation Format: Jessica A. Ayala-Cuesta, Yaya Chu, Keira Foley, Meijuan Tian, Kayleigh Klose, Alyssa S. Mendelowitz, Timothy P. Cripe, Dean A. Lee, Mitchell S. Cairo. Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3615.

Blockade of TGF-β signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo.

Blockade of TGF-β signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo.

Glial TGFβ activity promotes neuron survival in peripheral nerves.

Maintaining long, energetically demanding axons throughout the life of an animal is a major challenge for the nervous system. Specialized glia ensheathe axons and support their function and integrity throughout life, but glial support mechanisms remain poorly defined. Here, we identified a collection of secreted and transmembrane molecules required in glia for long-term axon survival in vivo. We showed that the majority of components of the TGFβ superfamily are required in glia for sensory neuron maintenance but not glial ensheathment of axons. In the absence of glial TGFβ signaling, neurons undergo age-dependent degeneration that can be rescued either by genetic blockade of Wallerian degeneration or caspase-dependent death. Blockade of glial TGFβ signaling results in increased ATP in glia that can be mimicked by enhancing glial mitochondrial biogenesis or suppressing glial monocarboxylate transporter function. We propose that glial TGFβ signaling supports axon survival and suppresses neurodegeneration through promoting glial metabolic support of neurons.

Blockade of TGF-β signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo.

TGF-β plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent the main cellular reservoir after long-term antiretroviral therapy (ART). We hypothesized that releasing cells from TGF-β-driven signaling would promote latency reversal. To test our hypothesis, we compared HIV-1 latency models with and without TGF-β and a TGF-β type 1 receptor inhibitor, galunisertib. We tested the effect of galunisertib in SIV-infected, ART-treated macaques by monitoring SIV-env expression via PET/CT using the 64Cu-DOTA-F(ab')2 p7D3 probe, along with plasma and tissue viral loads (VLs). Exogenous TGF-β reduced HIV-1 reactivation in U1 and ACH-2 models. Galunisertib increased HIV-1 latency reversal ex vivo and in PBMCs from HIV-1-infected, ART-treated, aviremic donors. In vivo, oral galunisertib promoted increased total standardized uptake values in PET/CT images in gut and lymph nodes of 5 out of 7 aviremic, long-term ART-treated, SIV-infected macaques. This increase correlated with an increase in SIV RNA in the gut. Two of the 7 animals also exhibited increases in plasma VLs. Higher anti-SIV T cell responses and antibody titers were detected after galunisertib treatment. In summary, our data suggest that blocking TGF-β signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.

Eliminating METTL1-mediated accumulation of PMN-MDSCs prevents hepatocellular carcinoma recurrence after radiofrequency ablation.

Radiofrequency ablation (RFA) is an important curative therapy in hepatocellular carcinoma (HCC), but recurrence rate remains as high as all the other HCC therapeutic modalities. Methyltransferase 1 (METTL1), an enzyme for m 7 G tRNA modification, was reported to promote HCC development. Here, we assessed the role of METTL1 in shaping the immunosuppressive tumor microenvironment after insufficient RFA (iRFA). By immunohistochemistry and multiplex immunofluorescence (mIF) staining, we showed that METTL1 expression was enhanced in post-RFA recurrent HCC, accompanied by increased CD11b + CD15 + polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and decreased CD8 + T cells. Mechanistically, heat-mediated METTL1 upregulation enhanced TGF-β2 translation to form the immunosuppressive environment by induction of myeloid-derived suppressor cell. Liver-specific overexpression or knockdown of Mettl1 significantly affected the accumulation of PMN-MDSCs and subsequently affected CD8 + T cell infiltration. Complete RFA successfully eliminated the tumor, whereas iRFA-treated mice exhibited enhanced tumor growth and metastasis with increased PMN-MDSC accumulation and decreased CD8 + T cells compared to sham surgery. Interrupting METTL1-TGF-β2-PMN-MDSC axis by anti-Ly6G antibody, or knockdown of hepatoma-intrinsic Mettl1 or Tgfb2 , or TGF-β signaling blockade significantly mitigated tumor progression induced by iRFA and restored CD8 + T cell population. Our study sheds light on the pivotal role of METTL1 in modulating an immunosuppressive microenvironment and demonstrated that interrupting METTL1-TGF-β2-PMN-MDSC axis could be a therapeutic strategy to restore antitumor immunity and prevent HCC recurrence after RFA treatment, meriting further clinical studies.

Blockade of TGF-β (Transforming Growth Factor Beta) Signaling by Deletion of Tgfbr2 in Smooth Muscle Cells of 11-Month-Old Mice Alters Aortic Structure and Causes Vasomotor Dysfunction-Brief Report.

To test the hypothesis that smooth muscle cell (SMC) TGF-β (transforming growth factor beta) signaling contributes to maintenance of aortic structure and function beyond the early postnatal period. We deleted the TBR2 (type 2 TGF-β receptor) in SMC of 11-month-old mice (genotype Acta2-CreERT2+/0Tgfbr2f/f, termed TBR2SMΔ) and compared their ascending aorta structure and vasomotor function to controls (Acta2-CreERT20/0Tgfbr2f/f, termed TBR2f/f). We confirmed loss of aortic SMC TBR2 by immunoblotting. Four weeks after SMC TBR2 loss, TBR2SMΔ mice did not have aortic rupture, ulceration, dissection, dilation, or evidence of medial hemorrhage. However, aortic medial area of TBR2SMΔ mice was increased by 27% (0.14±0.01 versus 0.11±0.01 mm2; P=0.01) and medial thickness was increased by 23% (40±1.9 versus 33±1.3 μm; P=0.004) compared with littermate controls. Wire myography performed on ascending aortic rings showed hypercontractility of TBR2SMΔ aortas to phenylephrine (Emax, 15.9±1.2 versus 10.8±0.7 mN; P=0.0003) and reduced relaxation and sensitivity to acetylcholine (Emax, 64±14% versus 96±2%; P=0.001; -logEC50, 6.9±0.1 versus 7.7±0.1; P=0.0001). Neither maximal relaxation nor sensitivity to sodium nitroprusside differed (Emax, 102±0.3% versus 101±0.3%; -logEC50, 8.0±0.04 versus 7.9±0.08; P>0.4 for both). Loss of TGF-β signaling in aortic SMC of 1-year-old mice does not cause early severe aortopathy or death; however, it causes mild structural and substantial physiological abnormalities. SMC TGF-β signaling plays an important role in maintaining aortic homeostasis in older mice. This role should be considered in the design of clinical studies that aim to prevent aortopathy by blocking SMC TGF-β signaling.

The Anti-fibrosis drug Pirfenidone modifies the immunosuppressive tumor microenvironment and prevents the progression of renal cell carcinoma by inhibiting tumor autocrine TGF-β

ABSTRACT Transforming growth factor-β (TGF-β) plays a critical role in regulating cell growth and differentiation. Epithelial to mesenchymal transition (EMT) induced by TGF-β promotes cancer cell migration, invasion, and proliferation. Pirfenidone (5-methyl-1-phenyl-2(1 H)-pyridone, PFD), an approved drug for treating pulmonary and renal fibrosis, is a potent TGF-β inhibitor and found reduced incidence of lung cancer and alleviated renal function decline. However, whether PFD plays a role in controlling renal cancer progression is largely unknown. In the present study, we demonstrated that high TGF-β1 expression was negatively associated with ten-year overall survival of patients with renal cancer. Functionally, blockade of TGF-β signaling with PFD significantly suppressed the progression of renal cancer in a murine model. Mechanistically, we revealed that PFD significantly decreased the expression and secretion of TGF-β both in vitro and in vivo tumor mouse model, which further prevented TGF-β-induced EMT and thus cell proliferation, migration, and invasion. Importantly, the downregulation of TGF-β upon PFD treatment shaped the immunosuppressive tumor microenvironment by limiting the recruitment of tumor-infiltrating MDSCs. Therefore, our study demonstrated that PFD prevents renal cancer progression by inhibiting TGF-β production of cancer cells and downstream signaling pathway, which might be presented as a therapeutic adjuvant for renal cancer.

Novel TGFβ Inhibitors Ameliorate Oral Squamous Cell Carcinoma Progression and Improve the Antitumor Immune Response of Anti-PD-L1 Immunotherapy.

TGFβ is a key regulator of oral squamous cell carcinoma (OSCC) progression, and its potential role as a therapeutic target has been investigated with a limited success. This study evaluates two novel TGFβ inhibitors as mono or combinatorial therapy with anti-PD-L1 antibodies (α-PD-L1 Ab) in a murine OSCC model. Immunocompetent C57BL/6 mice bearing malignant oral lesions induced by 4-nitroquinoline 1-oxide (4-NQO) were treated for 4 weeks with TGFβ inhibitors mRER (i.p., 50 μg/d) or mmTGFβ2-7m (10 μg/d delivered by osmotic pumps) alone or in combination with α-PD-L1 Abs (7× i.p. of 100 μg/72 h). Tumor progression and body weight were monitored. Levels of bioactive TGFβ in serum were quantified using a TGFβ bioassay. Tissues were analyzed by immunohistology and flow cytometry. Therapy with mRER or mmTGFβ2-7m reduced tumor burden (P < 0.05) and decreased body weight loss compared with controls. In inhibitor-treated mice, levels of TGFβ in tumor tissue and serum were reduced (P < 0.05), whereas they increased with tumor progression in controls. Both inhibitors enhanced CD8+ T-cell infiltration into tumors and mRER reduced levels of myeloid-derived suppressor cells (P < 0.001). In combination with α-PD-L1 Abs, tumor burden was not further reduced; however, mmTGFβ2-7m further reduced weight loss (P < 0.05). The collagen-rich stroma was reduced by using combinatorial TGFβ/PD-L1 therapies (P < 0.05), enabling an accelerated lymphocyte infiltration into tumor tissues. The blockade of TGFβ signaling by mRER or mmTGFβ2-7m ameliorated in vivo progression of established murine OSCC. The inhibitors promoted antitumor immune responses, alone and in combination with α-PD-L1 Abs.

TGFβ restricts expansion, survival, and function of T cells within the tuberculous granuloma

CD4 Tcell effector function is required for optimal containment of Mycobacterium tuberculosis (Mtb) infection. IFNɣ produced by CD4 Tcells is a key cytokine that contributes to protection. However, lung-infiltrating CD4 Tcells have a limited ability to produce IFNɣ, and IFNɣ plays a lesser protective role within the lung than at sites of Mtb dissemination. In a murine infection model, we observed that IFNɣ production by Mtb-specific CD4 Tcells is rapidly extinguished within the granuloma but not within unaffected lung regions, suggesting localized immunosuppression. We identified a signature of TGFβ signaling within granuloma-infiltrating Tcells in both mice and rhesus macaques. Selective blockade of TGFβ signaling in Tcells resulted in an accumulation of terminally differentiated effector CD4 Tcells, improved IFNɣ production within granulomas, and reduced bacterial burdens. These findings uncover a spatially localized immunosuppressive mechanism associated with Mtb infection and provide potential targets for host-directed therapy.

Pharmacologic Targeting of BET Proteins Attenuates Hyperuricemic Nephropathy in Rats.

Hyperuricemia is an independent risk factor for renal damage and promotes the progression of chronic kidney disease. In this study, we investigated the effect of I-BET151, a small-molecule inhibitor targeting the bromodomain and extraterminal (BET) proteins, on the development of hyperuricemic nephropathy (HN), and the mechanisms involved. Expression levels of bromodomain-containing protein 2 and 4, but not 3 were increased in the kidney of rats with HN; administration of I-BET151 effectively prevented renal dysfunction, decreased urine microalbumin, and attenuated renal fibrosis as indicated by reduced activation of renal interstitial fibroblasts and expression of fibronectin and collagen I in HN rats. Mechanistic studies show that I-BET151 treatment inhibited transition of renal epithelial cells to a mesenchymal cell type as evidenced by preservation of E-cadherin and reduction of vimentin expression. This was coincident with reduced expression of TGF-β1 and dephosphorylation of Smad3 and ERK1/2. I-BET151 was also effective in inhibiting phosphorylation of NF-κB, expression of multiple cytokines and chemokines, and infiltration of macrophages to the injured kidney. Although there were increased serum levels of uric acid and xanthine oxidase, an enzyme that catalyzes production of uric acid, and decreased expression of renal organic anion transporter 1 and 3 that promote urate excretion in the model of HN, and reduced expression levels of urine uric acid, I-BET151 treatment did not affect these responses. Collectively, our results indicate that I-BET151 alleviates HN by inhibiting epithelial to mesenchymal transition and inflammation in association with blockade of TGF-β, ERK1/2 and NF-κB signaling.

Autonomous TGFβ signaling induces phenotypic variation in human acute myeloid leukemia

Heterogeneity of leukemia stem cells (LSCs) is involved in their collective chemoresistance. To eradicate LSCs, it is necessary to understand the mechanisms underlying their heterogeneity. Here, we aimed to identify signals responsible for heterogeneity and variation of LSCs in human acute myeloid leukemia (AML). Monitoring expression levels of endothelial cell‐selective adhesion molecule (ESAM), a hematopoietic stem cell‐related marker, was useful to detect the plasticity of AML cells. While healthy human hematopoietic stem/progenitor cells robustly expressed ESAM, AML cells exhibited heterogeneous ESAM expression. Interestingly, ESAM− and ESAM+ leukemia cells obtained from AML patients were mutually interconvertible in culture. KG1a and CMK, human AML clones, also represented the heterogeneity in terms of ESAM expression. Single cell culture with ESAM− or ESAM+ AML clones recapitulated the phenotypic interconversion. The phenotypic alteration was regulated at the gene expression level, and RNA sequencing revealed activation of TGFβ signaling in these cells. AML cells secreted TGFβ1, which autonomously activated TGFβ pathway and induced their phenotypic variation. Surprisingly, TGFβ signaling blockade inhibited not only the variation but also the proliferation of AML cells. Therefore, autonomous activation of TGFβ signaling underlies the LSC heterogeneity, which may be a promising therapeutic target for AML.

Cancer immunotherapy via targeted TGF-β signalling blockade in TH cells.

Cancer arises from malignant cells that exist in dynamic multilevel interactions with the host tissue. Cancer therapies aiming to directly kill cancer cells, including oncogene-targeted therapy and immune-checkpoint therapy that revives tumour-reactive cytotoxic T lymphocytes, are effective in some patients1,2, but acquired resistance frequently develops3,4. An alternative therapeutic strategy aims to rectify the host tissue pathology, including abnormalities in the vasculature that foster cancer progression5,6; however, neutralization of proangiogenic factors such as vascular endothelial growth factor A (VEGFA) has had limited clinical benefits7,8. Here, following the finding that transforming growth factor-β (TGF-β) suppresses T helper 2 (TH2)-cell-mediated cancer immunity9, we show that blocking TGF-β signalling in CD4+ T cells remodels the tumour microenvironment and restrains cancer progression. In a mouse model of breast cancer resistant to immune-checkpoint or anti-VEGF therapies10,11, inducible genetic deletion of the TGF-β receptor II (TGFBR2) in CD4+ T cells suppressed tumour growth. For pharmacological blockade, we engineered a bispecific receptor decoy by attaching the TGF-β-neutralizing TGFBR2 extracellular domain to ibalizumab, a non-immunosuppressive CD4 antibody12,13, andnamed it CD4 TGF-β Trap (4T-Trap). Compared with a non-targeted TGF-β-Trap, 4T-Trap selectively inhibited TH cell TGF-β signalling in tumour-draining lymph nodes, causing reorganization of tumour vasculature and cancer cell death, a process dependent on the TH2 cytokine interleukin-4 (IL-4). Notably, the 4T-Trap-induced tumour tissue hypoxia led to increased VEGFA expression. VEGF inhibition enhanced the starvation-triggered cancer cell death and amplified the antitumour effect of 4T-Trap. Thus, targeted TGF-β signalling blockade in helper T cells elicits an effective tissue-level cancer defence response that can provide a basis for therapies directed towards the cancer environment.

Megakaryocyte TGFβ1 partitions erythropoiesis into immature progenitor/stem cells and maturing precursors

AbstractErythropoietin (EPO) provides the major survival signal to maturing erythroid precursors (EPs) and is essential for terminal erythropoiesis. Nonetheless, progenitor cells can irreversibly commit to an erythroid fate well before EPO acts, risking inefficiency if these progenitors are unneeded to maintain red blood cell (RBC) counts. We identified a new modular organization of erythropoiesis and, for the first time, demonstrate that the pre-EPO module is coupled to late EPO-dependent erythropoiesis by megakaryocyte (Mk) signals. Disrupting megakaryocytic transforming growth factor β1 (Tgfb1) disorganized hematopoiesis by expanding the pre-EPO pool of progenitor cells and consequently triggering significant apoptosis of EPO-dependent EPs. Similarly, pharmacologic blockade of TGFβ signaling in normal mice boosted the pre-EPO module, leading to apoptosis of EPO-sensitive EPs. Subsequent treatment with low-dose EPO triggered robust RBC production in both models. This work reveals modular regulation of erythropoiesis and offers a new strategy for overcoming chronic anemias.

CXCR4 Inhibition Enhances Efficacy of FLT3 Inhibitors in FLT3-Mutated AML Augmented by Suppressed TGF-b Signaling.

Given the proven importance of the CXCL12/CXCR4 axis in the stroma–acute myeloid leukemia (AML) interactions and the rapid emergence of resistance to FLT3 inhibitors, we investigated the efficacy and safety of a novel CXCR4 inhibitor, LY2510924, in combination with FLT3 inhibitors in preclinical models of AML with FLT3-ITD mutations (FLT3-ITD-AML). Quizartinib, a potent FLT3 inhibitor, induced apoptosis in FLT3-ITD-AML, while LY2510924 blocked surface CXCR4 without inducing apoptosis. LY2510924 significantly reversed stroma-mediated resistance against quizartinib mainly through the MAPK pathway. In mice with established FLT3-ITD-AML, LY2510924 induced durable mobilization and differentiation of leukemia cells, resulting in enhanced anti-leukemia effects when combined with quizartinib, whereas transient effects were seen on non-leukemic blood cells in immune-competent mice. Sequencing of the transcriptome of the leukemic cells surviving in vivo treatment with quizartinib and LY2510924 revealed that genes related to TGF-β signaling may confer resistance against the drug combination. In co-culture experiments of FLT3-ITD-AML and stromal cells, both silencing of TGF-β in stromal cells or TGF-β-receptor kinase inhibitor enhanced apoptosis by combined treatment. Disruption of the CXCL12/CXCR4 axis in FLT3-ITD-AML by LY2510924 and its negligible effects on normal immunocytes could safely enhance the potency of quizartinib, which may be further improved by blockade of TGF-β signaling.

Peripheral blood immune correlates associated with TGF-β inhibition (galunisertib) and stereotactic body radiotherapy (SBRT) in patients with advanced hepatocellular carcinoma (HCC).

4575 Background: TGF-β is a pleiotropic cytokine with immunosuppressive activity. In mouse models, blockade of TGF-β signaling enhances the activity of radiation and invokes T cell dependent anti-tumor immunity. We previously reported preliminary safety and efficacy results from a pilot study combining galunisertib (LY2157299), an oral TGF-β inhibitor, with SBRT for the treatment of patients with advanced HCC. Here, we investigate immunological mechanisms and potential biomarkers associated with therapeutic activity. Methods: Patients with advanced HCC who had progressed on or refused sorafenib were treated with galunisertib (150 mg PO BID) on days 1-14 of each 28-day cycle. SBRT (18 Gy) was delivered in a single dose between days 15-28 of cycle 1. Blood was collected at baseline, following two weeks of galunisertib and following SBRT for high-dimensional analysis using a 37-marker CyTOF panel. Patients were dichotomized based on best response as either progressor (PD) or non-progressor (PR+SD). The frequency of immune subsets was compared between groups. Results: Fifteen patients were enrolled and treated. One patient was not evaluable. The most common adverse event was grade 1 or 2 fatigue in 53% of patients. The only possibly-related grade 3 event was achalasia in one patient which coincided with disease progression. Two patients achieved a PR and six patients had SD (DCR 57%) with a median progression-free survival of 3.7 months and a median overall survival of 9.0 months. For most patients, regardless of outcome, galunisertib treatment was associated with a decrease in activated Ki67+ Treg cells. However, pre-treatment immune composition within the blood of progressors and non-progressors was distinct. At baseline, progressors had an increased frequency of naive-like CD8+ T cells, whereas non-progressors had an increased frequency of non-classical monocytes. After combination therapy, only non-progressors showed a significant increase in CD8+PD1+TIGIT+ T cells. Conclusions: Galunisertib combined with SBRT was well-tolerated with modest efficacy. Immune profiling of the blood revealed distinct pre- and post-treatment signatures that differentiated patients with progression versus non-progression. These findings show that the combination of anti-TGF-β therapy with radiation can mediate disease control and identify potential correlates of efficacy. Clinical trial information: NCT02906397 .