口服转化生长因子-β受体1抑制剂vactosertib通过靶向肿瘤增殖和增强抗肿瘤免疫力促进骨肉瘤消退。

IF 20.1 1区 医学 Q1 ONCOLOGY Cancer Communications Pub Date : 2024-07-06 DOI:10.1002/cac2.12589
Sung Hee Choi, Jay Thomas Myers, Suzanne Louise Tomchuck, Melissa Bonner, Saada Eid, Daniel Tyler Kingsley, Kristen Ashley VanHeyst, Seong-Jin Kim, Byung-Gyu Kim, Alex Yee-Chen Huang
{"title":"口服转化生长因子-β受体1抑制剂vactosertib通过靶向肿瘤增殖和增强抗肿瘤免疫力促进骨肉瘤消退。","authors":"Sung Hee Choi,&nbsp;Jay Thomas Myers,&nbsp;Suzanne Louise Tomchuck,&nbsp;Melissa Bonner,&nbsp;Saada Eid,&nbsp;Daniel Tyler Kingsley,&nbsp;Kristen Ashley VanHeyst,&nbsp;Seong-Jin Kim,&nbsp;Byung-Gyu Kim,&nbsp;Alex Yee-Chen Huang","doi":"10.1002/cac2.12589","DOIUrl":null,"url":null,"abstract":"<p>Osteosarcoma is an aggressive malignant bone sarcoma common among children, adolescents, and young adults. Approximately 20% of patients present with pulmonary metastasis, and an additional 40% develop pulmonary osteosarcoma later. The survival outcome in patients with recurrent osteosarcoma and pulmonary osteosarcoma has not improved over many decades [<span>1</span>]. Transforming growth factor-β (TGF-β) is a potent immunosuppressive molecule in the osteosarcoma tumor microenvironment (TME) known to suppress the function of cytotoxic T cells and natural killer (NK) cells and correlates with high-grade osteosarcoma and pulmonary osteosarcoma [<span>2</span>]. Vactosertib (TEW-7197) is a highly selective and potent small molecule inhibitor against Type 1 TGF-β Receptor (activin receptor-like kinase 5; ALK5) [<span>3</span>]. Vactosertib is orally available and has 10 times the potency of galunisertib (IC50 = 11×10<sup>−3</sup> µmol/L vs. 11×10<sup>−2</sup> µmol/L) when tested in 4T1 [<span>4</span>], and is well tolerated with a manageable safety profile in adults, representing an attractive option in osteosarcoma [<span>3</span>].</p><p>TGF-β1 levels correlate with overall survival in osteosarcoma patients (Figure 1A). Vactosertib directly suppressed mouse osteosarcoma and human osteosarcoma cell line growth in a dose-dependent manner, with an IC50 of 0.79-2.1 µmol/L (Figure 1B). Vactosertib (1 × 10<sup>−1</sup> µmol/L) completely suppressed the TGF-β signaling intermediate, p-Smad2, in mouse osteosarcoma and human osteosarcoma cells (Figure 1C). In contrast, other TGF-β1 inhibitors, SB431542 and galunisertib, exhibited an IC50 of 2.05 × 10<sup>3</sup> µmol/L and 12 µmol/L, respectively, and they were not able to suppress p-Smad2 at 1 × 10<sup>−1</sup> µmol/L in SAOS2 cells (Supplementary Figure S1A-B). Vactosertib (1 × 10<sup>−1</sup> µmol/L) treated SAOS2 cells displayed 35 upregulated and 72 downregulated genes, including decreased expression of Ephrin-2 (EFNB2), IL-11, and prostate transmembrane protein androgen induced1 (PMEPA1) which were all associated with osteosarcoma progression and metastasis (Supplementary Figure S2A) [<span>5</span>]. Gene Set Enrichment Analysis (GSEA) revealed 14 down-regulated gene sets, including Wnt Beta-catenin signaling, TGF-β1 and mammalian target of rapamycin complex 1 (mTORC1) signaling (Supplementary Figure S2B), with Myelocytomatosis (MYC) target genes among the most inhibited (Supplementary Figure S2B-C).</p><p>SAOS2 treated with TGF-β1 (5 ng/mL) alone most significantly increased c-Myc target genes, and vactosertib co-treatment with TGF-β1 significantly suppressed the same c-Myc target gene sets (Figure 1D). Expression of individual c-Myc target genes was independently confirmed using real-time reverse transcription-polymerase chain reaction (RT-PCR) (Supplementary Figure S2D). TGF-β1 (5 ng/ml) treatment alone also significantly increased c-Myc protein expression in SAOS2 cells, while a low dose of vactosertib (1 × 10<sup>−1</sup> µmol/L) completely abolished TGF-β1-induced c-Myc expression in SAOS2 cells (Figure 1C, Supplementary Figure S2E). This inhibition was extended beyond SAOS2 into other human osteosarcoma and mouse osteosarcoma cell lines (Figure 1C). Volcano plots identified <i>PMEPA1</i>, <i>LTBP1</i>, <i>IL-11</i> and <i>JUNB</i> as genes most significantly increased by TGF-β1 and suppressed by vactosertib co-treatment in SAOS2 cells (Supplementary Figure S2F-G). Previous studies have shown these genes to be involved in tumor progressions and metastasis, and JUNB has also been reported to bind the promoter of c-Myc and regulate its expression [<span>6</span>].</p><p>To test direct TGF-β inhibition of osteosarcoma growth in vivo, we administered vactosertib (50 mg/kg, 5 days/week, per os [p.o.]) starting 14 days after SAOS2 inoculation (subcutaneous [s.c.]) into NK-depleted Nude mice (Supplementary Figure S3A) and observed blunted tumor growth in vactosertib treated group (Supplementary Figure S3B). Similarly, improved survival rates, smaller tumor volume and reduced metastasis were observed in SAOS2-bearing NSG mice treated with vactosertib after almost 3 months (Supplementary Figure S3C-F), accompanied by a reduction in p-Smad2 (Figure S3G) and c-Myc mRNA expression in residual tumors in vivo (Supplementary Figure S3H). As c-Myc amplification has been reported in metastatic and chemo-resistant osteosarcoma [<span>7</span>], we tested vactosertib on 143B, a c-Myc amplified human osteosarcoma with high c-Myc at baseline (Supplementary Figure S3I). 143B growth was not inhibited by a wide dose range of vactosertib (10 × 10<sup>−3</sup> µmol/L to 10 × 10<sup>−6</sup> µmol/L) in vitro (Supplementary Figure S3J). Although vactosertib potently shut down pSmad2, ERK phosphorylation remained unchanged in 143B (Supplementary Figure S3I). Interestingly, vactosertib could not inhibit 143B tumor in NSG mice in vivo (Supplementary Figure S3K) but was able to do so in nude mice (Supplementary Figure S3L), suggesting a tumor-extrinsic effect of vactosertib such as through enhancement of NK cell function [<span>8</span>].</p><p>To assess the osteosarcoma-extrinsic effects of vactosertib on the immune landscape of the primary tumor sites, we employed a K7M2 model in BALB/c mice (Figure 1E). Vactosertib treatment (50 mg/kg, 5 days/week, p.o.) significantly inhibited K7M2 tumor growth (Figure 1F-G). While no statistically significant differences were observed in the percentage of the CD45, CD11b or MDSC (Ly6C<sup>lo</sup>Ly6G<sup>Hi</sup>, Ly6C<sup>Hi</sup>Ly6G<sup>−</sup>) cell populations (Supplementary Figure S3M), M2-like tumor-associated macrophages (TAMs) (CD11b<sup>+</sup>F4/80<sup>+</sup>Arg<sup>+</sup>PD-L1<sup>+</sup>) were significantly suppressed by vactosertib (Supplementary Figure S3M). Using a pulmonary osteosarcoma model where BALB/c mice were inoculated with 1×10<sup>6</sup> K7M2-Luc cells (i.v.) and treated with vactosertib via oral gavage starting 7 days later (Figure 1H), vactosertib-treated mice exhibited a dramatic inhibition of pulmonary osteosarcoma burden with a suppressed tumor c-Myc expression (Figure 1I-J). At a higher dose, vactosertib was efficacious in suppressing tumor growth even when starting late (3 weeks) at a higher tumor burden (Supplementary Figure S4A-C), accompanied by reduced lung metastasis (Supplementary Figure S4D-E). Interestingly, c-Myc expression in lung tissue was similar despite its clinical efficacy (Supplementary Figure S4F), implying a critical role for enhanced anti-tumor immunity in vivo.</p><p>Poor response to osteosarcoma therapy is correlated with low CD8<sup>+</sup> T cells and IFNγ expression [<span>9</span>]. To elucidate how vactosertib affects the in vivo immune landscape, we performed multiparametric flow cytometry (FACS) with tSNE analysis of lung tissues (Supplementary Figure S5A). The analysis showed vactosertib-exposed TME contained significantly more CD3<sup>+</sup>, IFN<sup>+</sup>CD8<sup>+</sup> and NK cells, along with a decreased prevalence of PD1<sup>+</sup>CD8<sup>+</sup> T cells, PD1<sup>+</sup>CD4<sup>+</sup> T cells (Figure 1K) and Foxp3<sup>+</sup>CD4<sup>+</sup> T cell subsets (data not shown). Vactosertib exposure resulted in the accumulation and deep infiltration of NK cells within the tumors while they were scattered and largely confined to the tumor periphery in vehicle controls (Supplementary Figure S6).</p><p>Examination of the myeloid cells (Supplementary Figure S5B) also showed a clear difference upon vactosertib treatment, with a suppression of M2-like TAMs expressing PD-L1<sup>+</sup>, CD206<sup>+</sup>PD-L1<sup>+</sup>, and Arg1<sup>+</sup> markers (Figure 1L). Similar to TAMs, vactosertib also diminished F4/80<sup>−</sup> CD11b<sup>+</sup>Ly6G<sup>−</sup>Ly6C<sup>+</sup> and CD11b<sup>+</sup>Ly6G<sup>+</sup>Ly6C<sup>+</sup> myeloid cells (data not shown), further supporting vactosertib as enhancing anti-tumor immunity in pulmonary osteosarcoma TME. Finally, we tested co-treatment with vactosertib and αPD-1/ αPD-L1 mAb for synergy against osteosarcoma in vivo (Supplementary Figure S7). Vactosertib alone inhibited osteosarcoma tumor growth as well as αPD-L1 mAb alone or in combination. Interestingly, in agreement with ongoing disappointing clinical observation in osteosarcoma patients receiving aPD-1 therapies [<span>10</span>], we did not observe a therapeutic efficacy with aPD-1 mAb in vivo. As vactosertib significantly reduced PD-1<sup>+</sup> T-cells and suppressed PD-L1<sup>+</sup> macrophages, the lack of synergistic effects of vactosertib and ICB may be explained by the reduction in the numbers of these cells. The exact mechanism(s) for this lack of clinical efficacy by targeting PD-1 in osteosarcoma await additional studies.</p><p>Based on our current study, a multi-continent (US, Europe, Asia), multi-center phase I/II clinical trial (NCT05588648) with vactosertib monotherapy for osteosarcoma is actively enrolling. The application of vactosertib as an adjuvant to additional cellular therapy and immune-modulating approaches for osteosarcoma and other cancers awaits thoughtful exploration, such as inclusion in protocols targeting early-stage and high-risk disease. Taken together, inhibition of TGF-β signaling could be an effective therapeutic strategy against pulmonary osteosarcoma through a multi-pronged approach that targets tumor intrinsic and extrinsic factors to achieve optimal immune-effector functions and maximal clinical response.</p><p>Sung Hee Choi and Alex Y. Huang conceptualized the project. Sung Hee Choi, Jay T. Myers, Suzanne L. Tomchuck, Melissa Bonner, Saada Eid, Daniel T. Kingsley, Kristen VanHeyst, and Byung-Gyu Kim performed experiments and analyzed data. Sung Hee Choi wrote the original draft of the manuscript. Jay T. Myers, Suzanne L. Tomchuck, Melissa Bonner, Saada Eid, Daniel T. Kingsley, Kristen VanHeyst, Seong-Jin Kim, Byung-Gyu Kim, and Alex Y. Huang reviewed and edited the manuscript. Alex Y. Huang acquired funding for the project. All authors have read and agreed to the published version of the manuscript.</p><p>S.J.K declares a personal financial interest as a shareholder in TheragenEtex and Medpacto Inc. and is an employee of Medpacto Inc..</p><p>This research was supported by the National Cancer Institute (R03CA273468, R03CA259901, P30CA043703, T32GM007250, T32CA059366, and K12CA076917), St. Baldrick's Foundation, Hyundai Hope-on-Wheels Scholar Hope Grant, Andrew McDonough B+ Foundation, Curing Kids Cancer, MIB Agents, Sarcoma Foundation of America, Sam Day Foundation, Children' ’s Cancer Research Fund, Center for Pediatric Immunotherapy at Rainbow, and a sponsored research agreement from MedPacto, Inc. who provided vactosertib for both in vitro and in vivo experiments.</p><p>All animal experiments were performed and monitored with strict adherence to protocols in accordance with institutional guidelines and with approval of the Institutional Animal Care and Use Committee (protocol # 2016-0067) at Case Western Reserve University School of Medicine and performed in accordance with the guidelines of the American Association for Accreditation of Laboratory Animal Care and the NIH.</p>","PeriodicalId":9495,"journal":{"name":"Cancer Communications","volume":null,"pages":null},"PeriodicalIF":20.1000,"publicationDate":"2024-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cac2.12589","citationCount":"0","resultStr":"{\"title\":\"Oral transforming growth factor-beta receptor 1 inhibitor vactosertib promotes osteosarcoma regression by targeting tumor proliferation and enhancing anti-tumor immunity\",\"authors\":\"Sung Hee Choi,&nbsp;Jay Thomas Myers,&nbsp;Suzanne Louise Tomchuck,&nbsp;Melissa Bonner,&nbsp;Saada Eid,&nbsp;Daniel Tyler Kingsley,&nbsp;Kristen Ashley VanHeyst,&nbsp;Seong-Jin Kim,&nbsp;Byung-Gyu Kim,&nbsp;Alex Yee-Chen Huang\",\"doi\":\"10.1002/cac2.12589\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Osteosarcoma is an aggressive malignant bone sarcoma common among children, adolescents, and young adults. Approximately 20% of patients present with pulmonary metastasis, and an additional 40% develop pulmonary osteosarcoma later. The survival outcome in patients with recurrent osteosarcoma and pulmonary osteosarcoma has not improved over many decades [<span>1</span>]. Transforming growth factor-β (TGF-β) is a potent immunosuppressive molecule in the osteosarcoma tumor microenvironment (TME) known to suppress the function of cytotoxic T cells and natural killer (NK) cells and correlates with high-grade osteosarcoma and pulmonary osteosarcoma [<span>2</span>]. Vactosertib (TEW-7197) is a highly selective and potent small molecule inhibitor against Type 1 TGF-β Receptor (activin receptor-like kinase 5; ALK5) [<span>3</span>]. Vactosertib is orally available and has 10 times the potency of galunisertib (IC50 = 11×10<sup>−3</sup> µmol/L vs. 11×10<sup>−2</sup> µmol/L) when tested in 4T1 [<span>4</span>], and is well tolerated with a manageable safety profile in adults, representing an attractive option in osteosarcoma [<span>3</span>].</p><p>TGF-β1 levels correlate with overall survival in osteosarcoma patients (Figure 1A). Vactosertib directly suppressed mouse osteosarcoma and human osteosarcoma cell line growth in a dose-dependent manner, with an IC50 of 0.79-2.1 µmol/L (Figure 1B). Vactosertib (1 × 10<sup>−1</sup> µmol/L) completely suppressed the TGF-β signaling intermediate, p-Smad2, in mouse osteosarcoma and human osteosarcoma cells (Figure 1C). In contrast, other TGF-β1 inhibitors, SB431542 and galunisertib, exhibited an IC50 of 2.05 × 10<sup>3</sup> µmol/L and 12 µmol/L, respectively, and they were not able to suppress p-Smad2 at 1 × 10<sup>−1</sup> µmol/L in SAOS2 cells (Supplementary Figure S1A-B). Vactosertib (1 × 10<sup>−1</sup> µmol/L) treated SAOS2 cells displayed 35 upregulated and 72 downregulated genes, including decreased expression of Ephrin-2 (EFNB2), IL-11, and prostate transmembrane protein androgen induced1 (PMEPA1) which were all associated with osteosarcoma progression and metastasis (Supplementary Figure S2A) [<span>5</span>]. Gene Set Enrichment Analysis (GSEA) revealed 14 down-regulated gene sets, including Wnt Beta-catenin signaling, TGF-β1 and mammalian target of rapamycin complex 1 (mTORC1) signaling (Supplementary Figure S2B), with Myelocytomatosis (MYC) target genes among the most inhibited (Supplementary Figure S2B-C).</p><p>SAOS2 treated with TGF-β1 (5 ng/mL) alone most significantly increased c-Myc target genes, and vactosertib co-treatment with TGF-β1 significantly suppressed the same c-Myc target gene sets (Figure 1D). Expression of individual c-Myc target genes was independently confirmed using real-time reverse transcription-polymerase chain reaction (RT-PCR) (Supplementary Figure S2D). TGF-β1 (5 ng/ml) treatment alone also significantly increased c-Myc protein expression in SAOS2 cells, while a low dose of vactosertib (1 × 10<sup>−1</sup> µmol/L) completely abolished TGF-β1-induced c-Myc expression in SAOS2 cells (Figure 1C, Supplementary Figure S2E). This inhibition was extended beyond SAOS2 into other human osteosarcoma and mouse osteosarcoma cell lines (Figure 1C). Volcano plots identified <i>PMEPA1</i>, <i>LTBP1</i>, <i>IL-11</i> and <i>JUNB</i> as genes most significantly increased by TGF-β1 and suppressed by vactosertib co-treatment in SAOS2 cells (Supplementary Figure S2F-G). Previous studies have shown these genes to be involved in tumor progressions and metastasis, and JUNB has also been reported to bind the promoter of c-Myc and regulate its expression [<span>6</span>].</p><p>To test direct TGF-β inhibition of osteosarcoma growth in vivo, we administered vactosertib (50 mg/kg, 5 days/week, per os [p.o.]) starting 14 days after SAOS2 inoculation (subcutaneous [s.c.]) into NK-depleted Nude mice (Supplementary Figure S3A) and observed blunted tumor growth in vactosertib treated group (Supplementary Figure S3B). Similarly, improved survival rates, smaller tumor volume and reduced metastasis were observed in SAOS2-bearing NSG mice treated with vactosertib after almost 3 months (Supplementary Figure S3C-F), accompanied by a reduction in p-Smad2 (Figure S3G) and c-Myc mRNA expression in residual tumors in vivo (Supplementary Figure S3H). As c-Myc amplification has been reported in metastatic and chemo-resistant osteosarcoma [<span>7</span>], we tested vactosertib on 143B, a c-Myc amplified human osteosarcoma with high c-Myc at baseline (Supplementary Figure S3I). 143B growth was not inhibited by a wide dose range of vactosertib (10 × 10<sup>−3</sup> µmol/L to 10 × 10<sup>−6</sup> µmol/L) in vitro (Supplementary Figure S3J). Although vactosertib potently shut down pSmad2, ERK phosphorylation remained unchanged in 143B (Supplementary Figure S3I). Interestingly, vactosertib could not inhibit 143B tumor in NSG mice in vivo (Supplementary Figure S3K) but was able to do so in nude mice (Supplementary Figure S3L), suggesting a tumor-extrinsic effect of vactosertib such as through enhancement of NK cell function [<span>8</span>].</p><p>To assess the osteosarcoma-extrinsic effects of vactosertib on the immune landscape of the primary tumor sites, we employed a K7M2 model in BALB/c mice (Figure 1E). Vactosertib treatment (50 mg/kg, 5 days/week, p.o.) significantly inhibited K7M2 tumor growth (Figure 1F-G). While no statistically significant differences were observed in the percentage of the CD45, CD11b or MDSC (Ly6C<sup>lo</sup>Ly6G<sup>Hi</sup>, Ly6C<sup>Hi</sup>Ly6G<sup>−</sup>) cell populations (Supplementary Figure S3M), M2-like tumor-associated macrophages (TAMs) (CD11b<sup>+</sup>F4/80<sup>+</sup>Arg<sup>+</sup>PD-L1<sup>+</sup>) were significantly suppressed by vactosertib (Supplementary Figure S3M). Using a pulmonary osteosarcoma model where BALB/c mice were inoculated with 1×10<sup>6</sup> K7M2-Luc cells (i.v.) and treated with vactosertib via oral gavage starting 7 days later (Figure 1H), vactosertib-treated mice exhibited a dramatic inhibition of pulmonary osteosarcoma burden with a suppressed tumor c-Myc expression (Figure 1I-J). At a higher dose, vactosertib was efficacious in suppressing tumor growth even when starting late (3 weeks) at a higher tumor burden (Supplementary Figure S4A-C), accompanied by reduced lung metastasis (Supplementary Figure S4D-E). Interestingly, c-Myc expression in lung tissue was similar despite its clinical efficacy (Supplementary Figure S4F), implying a critical role for enhanced anti-tumor immunity in vivo.</p><p>Poor response to osteosarcoma therapy is correlated with low CD8<sup>+</sup> T cells and IFNγ expression [<span>9</span>]. To elucidate how vactosertib affects the in vivo immune landscape, we performed multiparametric flow cytometry (FACS) with tSNE analysis of lung tissues (Supplementary Figure S5A). The analysis showed vactosertib-exposed TME contained significantly more CD3<sup>+</sup>, IFN<sup>+</sup>CD8<sup>+</sup> and NK cells, along with a decreased prevalence of PD1<sup>+</sup>CD8<sup>+</sup> T cells, PD1<sup>+</sup>CD4<sup>+</sup> T cells (Figure 1K) and Foxp3<sup>+</sup>CD4<sup>+</sup> T cell subsets (data not shown). Vactosertib exposure resulted in the accumulation and deep infiltration of NK cells within the tumors while they were scattered and largely confined to the tumor periphery in vehicle controls (Supplementary Figure S6).</p><p>Examination of the myeloid cells (Supplementary Figure S5B) also showed a clear difference upon vactosertib treatment, with a suppression of M2-like TAMs expressing PD-L1<sup>+</sup>, CD206<sup>+</sup>PD-L1<sup>+</sup>, and Arg1<sup>+</sup> markers (Figure 1L). Similar to TAMs, vactosertib also diminished F4/80<sup>−</sup> CD11b<sup>+</sup>Ly6G<sup>−</sup>Ly6C<sup>+</sup> and CD11b<sup>+</sup>Ly6G<sup>+</sup>Ly6C<sup>+</sup> myeloid cells (data not shown), further supporting vactosertib as enhancing anti-tumor immunity in pulmonary osteosarcoma TME. Finally, we tested co-treatment with vactosertib and αPD-1/ αPD-L1 mAb for synergy against osteosarcoma in vivo (Supplementary Figure S7). Vactosertib alone inhibited osteosarcoma tumor growth as well as αPD-L1 mAb alone or in combination. Interestingly, in agreement with ongoing disappointing clinical observation in osteosarcoma patients receiving aPD-1 therapies [<span>10</span>], we did not observe a therapeutic efficacy with aPD-1 mAb in vivo. As vactosertib significantly reduced PD-1<sup>+</sup> T-cells and suppressed PD-L1<sup>+</sup> macrophages, the lack of synergistic effects of vactosertib and ICB may be explained by the reduction in the numbers of these cells. The exact mechanism(s) for this lack of clinical efficacy by targeting PD-1 in osteosarcoma await additional studies.</p><p>Based on our current study, a multi-continent (US, Europe, Asia), multi-center phase I/II clinical trial (NCT05588648) with vactosertib monotherapy for osteosarcoma is actively enrolling. The application of vactosertib as an adjuvant to additional cellular therapy and immune-modulating approaches for osteosarcoma and other cancers awaits thoughtful exploration, such as inclusion in protocols targeting early-stage and high-risk disease. Taken together, inhibition of TGF-β signaling could be an effective therapeutic strategy against pulmonary osteosarcoma through a multi-pronged approach that targets tumor intrinsic and extrinsic factors to achieve optimal immune-effector functions and maximal clinical response.</p><p>Sung Hee Choi and Alex Y. Huang conceptualized the project. Sung Hee Choi, Jay T. Myers, Suzanne L. Tomchuck, Melissa Bonner, Saada Eid, Daniel T. Kingsley, Kristen VanHeyst, and Byung-Gyu Kim performed experiments and analyzed data. Sung Hee Choi wrote the original draft of the manuscript. Jay T. Myers, Suzanne L. Tomchuck, Melissa Bonner, Saada Eid, Daniel T. Kingsley, Kristen VanHeyst, Seong-Jin Kim, Byung-Gyu Kim, and Alex Y. Huang reviewed and edited the manuscript. Alex Y. Huang acquired funding for the project. All authors have read and agreed to the published version of the manuscript.</p><p>S.J.K declares a personal financial interest as a shareholder in TheragenEtex and Medpacto Inc. and is an employee of Medpacto Inc..</p><p>This research was supported by the National Cancer Institute (R03CA273468, R03CA259901, P30CA043703, T32GM007250, T32CA059366, and K12CA076917), St. Baldrick's Foundation, Hyundai Hope-on-Wheels Scholar Hope Grant, Andrew McDonough B+ Foundation, Curing Kids Cancer, MIB Agents, Sarcoma Foundation of America, Sam Day Foundation, Children' ’s Cancer Research Fund, Center for Pediatric Immunotherapy at Rainbow, and a sponsored research agreement from MedPacto, Inc. who provided vactosertib for both in vitro and in vivo experiments.</p><p>All animal experiments were performed and monitored with strict adherence to protocols in accordance with institutional guidelines and with approval of the Institutional Animal Care and Use Committee (protocol # 2016-0067) at Case Western Reserve University School of Medicine and performed in accordance with the guidelines of the American Association for Accreditation of Laboratory Animal Care and the NIH.</p>\",\"PeriodicalId\":9495,\"journal\":{\"name\":\"Cancer Communications\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":20.1000,\"publicationDate\":\"2024-07-06\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cac2.12589\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cancer Communications\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12589\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"ONCOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cancer Communications","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12589","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ONCOLOGY","Score":null,"Total":0}
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摘要

骨肉瘤是一种侵袭性恶性骨肉瘤,常见于儿童、青少年和年轻人。约 20% 的患者会出现肺转移,另有 40% 的患者会在后期发展为肺骨肉瘤。几十年来,复发性骨肉瘤和肺骨肉瘤患者的生存率一直没有提高[1]。转化生长因子-β(TGF-β)是骨肉瘤肿瘤微环境(TME)中的一种强效免疫抑制分子,已知可抑制细胞毒性T细胞和自然杀伤(NK)细胞的功能,并与高级别骨肉瘤和肺骨肉瘤相关[2]。Vactosertib(TEW-7197)是一种针对1型TGF-β受体(活化素受体样激酶5;ALK5)的高选择性强效小分子抑制剂[3]。Vactosertib可口服,在4T1中的测试结果是galunisertib的10倍(IC50 = 11×10-3 µmol/L vs. 11×10-2 µmol/L)[4],在成人中耐受性良好,安全性可控,是骨肉瘤治疗中一个有吸引力的选择[3]。Vactosertib 可直接抑制小鼠骨肉瘤和人骨肉瘤细胞系的生长,其作用呈剂量依赖性,IC50 为 0.79-2.1 µmol/L(图 1B)。Vactosertib(1×10-1 µmol/L)完全抑制了小鼠骨肉瘤和人骨肉瘤细胞中的TGF-β信号转导中间体p-Smad2(图1C)。相比之下,其他TGF-β1抑制剂SB431542和galunisertib的IC50分别为2.05×103 µmol/L和12 µmol/L,它们在1×10-1 µmol/L时无法抑制SAOS2细胞中的p-Smad2(补充图S1A-B)。Vactosertib(1 × 10-1 µmol/L)处理的SAOS2细胞显示了35个上调基因和72个下调基因,包括Ephrin-2(EFNB2)、IL-11和前列腺跨膜蛋白雄激素诱导1(PMEPA1)的表达减少,而这些基因都与骨肉瘤的进展和转移有关(补充图S2A)[5]。基因组富集分析(Gene Set Enrichment Analysis,GSEA)发现了 14 个下调基因组,包括 Wnt Beta-catenin 信号转导、TGF-β1 和哺乳动物雷帕霉素靶复合物 1(mTORC1)信号转导(补充图 S2B),其中骨髓细胞瘤病(MYC)靶基因受到的抑制最大(补充图 S2B-C)。SAOS2 单独与 TGF-β1 (5 ng/mL)处理时,c-Myc 靶基因的增加最为显著,而 vactosertib 与 TGF-β1 联合处理时,c-Myc 靶基因集的增加显著受抑制(图 1D)。使用实时反转录聚合酶链反应(RT-PCR)独立证实了各个c-Myc靶基因的表达(补充图S2D)。单独处理 TGF-β1(5 ng/ml)也会显著增加 SAOS2 细胞中 c-Myc 蛋白的表达,而低剂量的 vactosertib(1 × 10-1 µmol/L)会完全抑制 TGF-β1 诱导的 SAOS2 细胞中 c-Myc 的表达(图 1C,补充图 S2E)。这种抑制作用已从 SAOS2 扩展到其他人类骨肉瘤和小鼠骨肉瘤细胞系(图 1C)。火山图发现,在 SAOS2 细胞中,PMEPA1、LTBP1、IL-11 和 JUNB 是受 TGF-β1 影响最显著增加的基因,而与 vactosertib 联合处理则会抑制这些基因(补充图 S2F-G)。先前的研究表明,这些基因参与了肿瘤的进展和转移,JUNB也被报道与c-Myc的启动子结合并调控其表达[6]。为了测试TGF-β对骨肉瘤体内生长的直接抑制作用,我们在SAOS2细胞生长14天后开始给予vactosertib(50 mg/kg,5天/周,per os [p。o.]),并观察到vactosertib治疗组的肿瘤生长减弱(补充图S3B)。同样,用vactosertib治疗SAOS2的NSG小鼠近3个月后,观察到生存率提高、肿瘤体积缩小和转移减少(补充图S3C-F),同时体内残余肿瘤中p-Smad2(图S3G)和c-Myc mRNA表达减少(补充图S3H)。据报道,c-Myc扩增存在于转移性和化疗耐药的骨肉瘤中[7],因此我们在基线c-Myc较高的c-Myc扩增人骨肉瘤143B上测试了vactosertib(补充图S3I)。143B 的生长在体外没有受到较大剂量范围的 vactosertib(10 × 10-3 µmol/L 至 10 × 10-6 µmol/L)的抑制(补充图 S3J)。虽然vactosertib有效地关闭了pSmad2,但ERK磷酸化在143B中保持不变(补充图S3I)。有趣的是,vactosertib不能抑制NSG小鼠体内的143B肿瘤(补图S3K),但却能抑制裸鼠体内的143B肿瘤(补图S3L),这表明vactosertib具有肿瘤外效应,例如通过增强NK细胞的功能[8]。
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Oral transforming growth factor-beta receptor 1 inhibitor vactosertib promotes osteosarcoma regression by targeting tumor proliferation and enhancing anti-tumor immunity

Osteosarcoma is an aggressive malignant bone sarcoma common among children, adolescents, and young adults. Approximately 20% of patients present with pulmonary metastasis, and an additional 40% develop pulmonary osteosarcoma later. The survival outcome in patients with recurrent osteosarcoma and pulmonary osteosarcoma has not improved over many decades [1]. Transforming growth factor-β (TGF-β) is a potent immunosuppressive molecule in the osteosarcoma tumor microenvironment (TME) known to suppress the function of cytotoxic T cells and natural killer (NK) cells and correlates with high-grade osteosarcoma and pulmonary osteosarcoma [2]. Vactosertib (TEW-7197) is a highly selective and potent small molecule inhibitor against Type 1 TGF-β Receptor (activin receptor-like kinase 5; ALK5) [3]. Vactosertib is orally available and has 10 times the potency of galunisertib (IC50 = 11×10−3 µmol/L vs. 11×10−2 µmol/L) when tested in 4T1 [4], and is well tolerated with a manageable safety profile in adults, representing an attractive option in osteosarcoma [3].

TGF-β1 levels correlate with overall survival in osteosarcoma patients (Figure 1A). Vactosertib directly suppressed mouse osteosarcoma and human osteosarcoma cell line growth in a dose-dependent manner, with an IC50 of 0.79-2.1 µmol/L (Figure 1B). Vactosertib (1 × 10−1 µmol/L) completely suppressed the TGF-β signaling intermediate, p-Smad2, in mouse osteosarcoma and human osteosarcoma cells (Figure 1C). In contrast, other TGF-β1 inhibitors, SB431542 and galunisertib, exhibited an IC50 of 2.05 × 103 µmol/L and 12 µmol/L, respectively, and they were not able to suppress p-Smad2 at 1 × 10−1 µmol/L in SAOS2 cells (Supplementary Figure S1A-B). Vactosertib (1 × 10−1 µmol/L) treated SAOS2 cells displayed 35 upregulated and 72 downregulated genes, including decreased expression of Ephrin-2 (EFNB2), IL-11, and prostate transmembrane protein androgen induced1 (PMEPA1) which were all associated with osteosarcoma progression and metastasis (Supplementary Figure S2A) [5]. Gene Set Enrichment Analysis (GSEA) revealed 14 down-regulated gene sets, including Wnt Beta-catenin signaling, TGF-β1 and mammalian target of rapamycin complex 1 (mTORC1) signaling (Supplementary Figure S2B), with Myelocytomatosis (MYC) target genes among the most inhibited (Supplementary Figure S2B-C).

SAOS2 treated with TGF-β1 (5 ng/mL) alone most significantly increased c-Myc target genes, and vactosertib co-treatment with TGF-β1 significantly suppressed the same c-Myc target gene sets (Figure 1D). Expression of individual c-Myc target genes was independently confirmed using real-time reverse transcription-polymerase chain reaction (RT-PCR) (Supplementary Figure S2D). TGF-β1 (5 ng/ml) treatment alone also significantly increased c-Myc protein expression in SAOS2 cells, while a low dose of vactosertib (1 × 10−1 µmol/L) completely abolished TGF-β1-induced c-Myc expression in SAOS2 cells (Figure 1C, Supplementary Figure S2E). This inhibition was extended beyond SAOS2 into other human osteosarcoma and mouse osteosarcoma cell lines (Figure 1C). Volcano plots identified PMEPA1, LTBP1, IL-11 and JUNB as genes most significantly increased by TGF-β1 and suppressed by vactosertib co-treatment in SAOS2 cells (Supplementary Figure S2F-G). Previous studies have shown these genes to be involved in tumor progressions and metastasis, and JUNB has also been reported to bind the promoter of c-Myc and regulate its expression [6].

To test direct TGF-β inhibition of osteosarcoma growth in vivo, we administered vactosertib (50 mg/kg, 5 days/week, per os [p.o.]) starting 14 days after SAOS2 inoculation (subcutaneous [s.c.]) into NK-depleted Nude mice (Supplementary Figure S3A) and observed blunted tumor growth in vactosertib treated group (Supplementary Figure S3B). Similarly, improved survival rates, smaller tumor volume and reduced metastasis were observed in SAOS2-bearing NSG mice treated with vactosertib after almost 3 months (Supplementary Figure S3C-F), accompanied by a reduction in p-Smad2 (Figure S3G) and c-Myc mRNA expression in residual tumors in vivo (Supplementary Figure S3H). As c-Myc amplification has been reported in metastatic and chemo-resistant osteosarcoma [7], we tested vactosertib on 143B, a c-Myc amplified human osteosarcoma with high c-Myc at baseline (Supplementary Figure S3I). 143B growth was not inhibited by a wide dose range of vactosertib (10 × 10−3 µmol/L to 10 × 10−6 µmol/L) in vitro (Supplementary Figure S3J). Although vactosertib potently shut down pSmad2, ERK phosphorylation remained unchanged in 143B (Supplementary Figure S3I). Interestingly, vactosertib could not inhibit 143B tumor in NSG mice in vivo (Supplementary Figure S3K) but was able to do so in nude mice (Supplementary Figure S3L), suggesting a tumor-extrinsic effect of vactosertib such as through enhancement of NK cell function [8].

To assess the osteosarcoma-extrinsic effects of vactosertib on the immune landscape of the primary tumor sites, we employed a K7M2 model in BALB/c mice (Figure 1E). Vactosertib treatment (50 mg/kg, 5 days/week, p.o.) significantly inhibited K7M2 tumor growth (Figure 1F-G). While no statistically significant differences were observed in the percentage of the CD45, CD11b or MDSC (Ly6CloLy6GHi, Ly6CHiLy6G) cell populations (Supplementary Figure S3M), M2-like tumor-associated macrophages (TAMs) (CD11b+F4/80+Arg+PD-L1+) were significantly suppressed by vactosertib (Supplementary Figure S3M). Using a pulmonary osteosarcoma model where BALB/c mice were inoculated with 1×106 K7M2-Luc cells (i.v.) and treated with vactosertib via oral gavage starting 7 days later (Figure 1H), vactosertib-treated mice exhibited a dramatic inhibition of pulmonary osteosarcoma burden with a suppressed tumor c-Myc expression (Figure 1I-J). At a higher dose, vactosertib was efficacious in suppressing tumor growth even when starting late (3 weeks) at a higher tumor burden (Supplementary Figure S4A-C), accompanied by reduced lung metastasis (Supplementary Figure S4D-E). Interestingly, c-Myc expression in lung tissue was similar despite its clinical efficacy (Supplementary Figure S4F), implying a critical role for enhanced anti-tumor immunity in vivo.

Poor response to osteosarcoma therapy is correlated with low CD8+ T cells and IFNγ expression [9]. To elucidate how vactosertib affects the in vivo immune landscape, we performed multiparametric flow cytometry (FACS) with tSNE analysis of lung tissues (Supplementary Figure S5A). The analysis showed vactosertib-exposed TME contained significantly more CD3+, IFN+CD8+ and NK cells, along with a decreased prevalence of PD1+CD8+ T cells, PD1+CD4+ T cells (Figure 1K) and Foxp3+CD4+ T cell subsets (data not shown). Vactosertib exposure resulted in the accumulation and deep infiltration of NK cells within the tumors while they were scattered and largely confined to the tumor periphery in vehicle controls (Supplementary Figure S6).

Examination of the myeloid cells (Supplementary Figure S5B) also showed a clear difference upon vactosertib treatment, with a suppression of M2-like TAMs expressing PD-L1+, CD206+PD-L1+, and Arg1+ markers (Figure 1L). Similar to TAMs, vactosertib also diminished F4/80 CD11b+Ly6GLy6C+ and CD11b+Ly6G+Ly6C+ myeloid cells (data not shown), further supporting vactosertib as enhancing anti-tumor immunity in pulmonary osteosarcoma TME. Finally, we tested co-treatment with vactosertib and αPD-1/ αPD-L1 mAb for synergy against osteosarcoma in vivo (Supplementary Figure S7). Vactosertib alone inhibited osteosarcoma tumor growth as well as αPD-L1 mAb alone or in combination. Interestingly, in agreement with ongoing disappointing clinical observation in osteosarcoma patients receiving aPD-1 therapies [10], we did not observe a therapeutic efficacy with aPD-1 mAb in vivo. As vactosertib significantly reduced PD-1+ T-cells and suppressed PD-L1+ macrophages, the lack of synergistic effects of vactosertib and ICB may be explained by the reduction in the numbers of these cells. The exact mechanism(s) for this lack of clinical efficacy by targeting PD-1 in osteosarcoma await additional studies.

Based on our current study, a multi-continent (US, Europe, Asia), multi-center phase I/II clinical trial (NCT05588648) with vactosertib monotherapy for osteosarcoma is actively enrolling. The application of vactosertib as an adjuvant to additional cellular therapy and immune-modulating approaches for osteosarcoma and other cancers awaits thoughtful exploration, such as inclusion in protocols targeting early-stage and high-risk disease. Taken together, inhibition of TGF-β signaling could be an effective therapeutic strategy against pulmonary osteosarcoma through a multi-pronged approach that targets tumor intrinsic and extrinsic factors to achieve optimal immune-effector functions and maximal clinical response.

Sung Hee Choi and Alex Y. Huang conceptualized the project. Sung Hee Choi, Jay T. Myers, Suzanne L. Tomchuck, Melissa Bonner, Saada Eid, Daniel T. Kingsley, Kristen VanHeyst, and Byung-Gyu Kim performed experiments and analyzed data. Sung Hee Choi wrote the original draft of the manuscript. Jay T. Myers, Suzanne L. Tomchuck, Melissa Bonner, Saada Eid, Daniel T. Kingsley, Kristen VanHeyst, Seong-Jin Kim, Byung-Gyu Kim, and Alex Y. Huang reviewed and edited the manuscript. Alex Y. Huang acquired funding for the project. All authors have read and agreed to the published version of the manuscript.

S.J.K declares a personal financial interest as a shareholder in TheragenEtex and Medpacto Inc. and is an employee of Medpacto Inc..

This research was supported by the National Cancer Institute (R03CA273468, R03CA259901, P30CA043703, T32GM007250, T32CA059366, and K12CA076917), St. Baldrick's Foundation, Hyundai Hope-on-Wheels Scholar Hope Grant, Andrew McDonough B+ Foundation, Curing Kids Cancer, MIB Agents, Sarcoma Foundation of America, Sam Day Foundation, Children' ’s Cancer Research Fund, Center for Pediatric Immunotherapy at Rainbow, and a sponsored research agreement from MedPacto, Inc. who provided vactosertib for both in vitro and in vivo experiments.

All animal experiments were performed and monitored with strict adherence to protocols in accordance with institutional guidelines and with approval of the Institutional Animal Care and Use Committee (protocol # 2016-0067) at Case Western Reserve University School of Medicine and performed in accordance with the guidelines of the American Association for Accreditation of Laboratory Animal Care and the NIH.

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来源期刊
Cancer Communications
Cancer Communications Biochemistry, Genetics and Molecular Biology-Cancer Research
CiteScore
25.50
自引率
4.30%
发文量
153
审稿时长
4 weeks
期刊介绍: Cancer Communications is an open access, peer-reviewed online journal that encompasses basic, clinical, and translational cancer research. The journal welcomes submissions concerning clinical trials, epidemiology, molecular and cellular biology, and genetics.
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