Blockade of the TGF ss signaling pathway has emerged from preclinical studies as a potential treatment to enhance the efficacy of immune checkpoint inhibition in advanced colorectal cancer (CRC) and several other types of cancer. However, clinical translation of first-generation inhibitors has shown little success. Here, we report the synthesis and characterization of HYL001, a potent inhibitor of TGF ss receptor 1 (ALK5), that is approximately 9 times more efficacious than the structurally related compound galunisertib, while maintaining a favorable safety profile. HYL001 in combination with immune checkpoint blockade (anti-PD1) eradicates liver metastases generated in mice by microsatellite stable, aggressive colorectal cancer tumors at doses where galunisertib is ineffective.

Blockade of the TGFβ signalling pathway has emerged from preclinical studies as a potential treatment to enhance the efficacy of immune checkpoint inhibition in advanced colorectal cancer (CRC) and several other types of cancer. However, clinical translation of first-generation inhibitors has known little success. Here, we report the synthesis and characterization of HYL001, a potent inhibitor of TGFβ receptor 1 (ALK5), that is approximately 9 times more efficacious than the structurally related compound galunisertib, while maintaining a favourable safety profile. HYL001 in combination with immune checkpoint blockade (anti-PD1) eradicates liver metastases generated in mice by microsatellite stable, aggressive colorectal cancer tumours at doses where galunisertib is ineffective. ![Figure][1] ### Competing Interest Statement DT, DB, JM, AR, and EB hold a patent on the synthesis and use of HYL001. E.B. is author in a patent describing bispecific antibodies to target cancer stem cells. The laboratory of E.B. has received research funding from MERUS and INCYTE. E.B. has received honoraria for consulting from Genentech. [1]: pending:yes

Most patients with colorectal cancer die as a result of the disease spreading to other organs. However, no prevalent mutations have been associated with metastatic colorectal cancers. Instead, particular features of the tumour microenvironment, such as lack of T-cell infiltration, low type 1 T-helper cell (TH1) activity and reduced immune cytotoxicity or increased TGFβ levels predict adverse outcomes in patients with colorectal cancer. Here we analyse the interplay between genetic alterations and the tumour microenvironment by crossing mice bearing conditional alleles of four main colorectal cancer mutations in intestinal stem cells. Quadruple-mutant mice developed metastatic intestinal tumours that display key hallmarks of human microsatellite-stable colorectal cancers, including low mutational burden, T-cell exclusion and TGFβ-activated stroma. Inhibition of the PD-1-PD-L1 immune checkpoint provoked a limited response in this model system. By contrast, inhibition of TGFβ unleashed a potent and enduring cytotoxic T-cell response against tumour cells that prevented metastasis. In mice with progressive liver metastatic disease, blockade of TGFβ signalling rendered tumours susceptible to anti-PD-1-PD-L1 therapy. Our data show that increased TGFβ in the tumour microenvironment represents a primary mechanism of immune evasion that promotes T-cell exclusion and blocks acquisition of the TH1-effector phenotype. Immunotherapies directed against TGFβ signalling may therefore have broad applications in treating patients with advanced colorectal cancer.

Recent molecular classifications of colorectal cancer (CRC) based on global gene expression profiles have defined subtypes displaying resistance to therapy and poor prognosis. Upon evaluation of these classification systems, we discovered that their predictive power arises from genes expressed by stromal cells rather than epithelial tumor cells. Bioinformatic and immunohistochemical analyses identify stromal markers that associate robustly with disease relapse across the various classifications. Functional studies indicate that cancer-associated fibroblasts (CAFs) increase the frequency of tumor-initiating cells, an effect that is dramatically enhanced by transforming growth factor (TGF)-β signaling. Likewise, we find that all poor-prognosis CRC subtypes share a gene program induced by TGF-β in tumor stromal cells. Using patient-derived tumor organoids and xenografts, we show that the use of TGF-β signaling inhibitors to block the cross-talk between cancer cells and the microenvironment halts disease progression.

We sought to understand the mechanisms behind the potent effect of stromal TGF-beta program on the capacity of colorectal cancer (CRC) cells to initiate metastasis. We discovered that mice subcutaneous tumors and metastases generated in the context of a TGF-beta activated microenvironment displayed prominent accumulation of p-STAT3 in CRC cells compared with those derived from control cells. STAT3 signaling depended on GP130 as shown by strong reduction of epithelial p STAT3 levels upon GP130 shRNA-mediated knockdown in CRC cells.

Metastasis depends on a gene program expressed by the tumor microenvironment upon TGF-beta stimulation. CRC (Colorectal cancer) cell lines did not induce robust stromal TGF-beta responses when injected into nude mice as shown by lack of p-SMAD2 accumulation in tumor-associated stromal cells. To enforce high TGF-beta signaling in xenografts, we engineered CRC cell lines to secrete active TGF-beta. Subcutaneous tumors obtained from HT29-M6TGF-β, KM12L4aTGF-β cells and SW48TGF-β cells contained abundant p-SMAD2+ stromal cells.

The stereo divergent ring-opening of 2-phenyl oxazaphospholidines with alkyl lithium reagents is reported. N-H oxazaphospholidines derived from both (+)-cis-1-amino-2-indanol and (-)-norephedrine provide inversion products in a highly stereoselective process. In contrast, N-Me oxazaphospholidines yield ring opening products with retention of configuration at the P center, as previously reported by Juge and co-workers. As a result, from a single amino alcohol auxiliary, both enantiomers of key P-stereogenic intermediates could be synthesized Theoretical studies of ring opening with model oxazaphospholidines at the DFT level have elucidated the streochemical course of this process. N-H substrates react in a single step via preferential backside S(N)2@P substitution with inversion at phosphorus. N-methylated substrates react preferentially via a two-step frontside S(N)2@P, yielding a ring-opened product in which the nucleophilic methyl binds to P with retention of configuration. DFT calculations have shown that the BH3 unit is a potent directing group to which the methyl lithium reagent coordinates via Li in all the reactions studied.

A large proportion of colorectal cancers (CRCs) display mutational inactivation of the TGF-β pathway, yet, paradoxically, they are characterized by elevated TGF-β production. Here, we unveil a prometastatic program induced by TGF-β in the microenvironment that associates with a high risk of CRC relapse upon treatment. The activity of TGF-β on stromal cells increases the efficiency of organ colonization by CRC cells, whereas mice treated with a pharmacological inhibitor of TGFBR1 are resilient to metastasis formation. Secretion of IL11 by TGF-β-stimulated cancer-associated fibroblasts (CAFs) triggers GP130/STAT3 signaling in tumor cells. This crosstalk confers a survival advantage to metastatic cells. The dependency on the TGF-β stromal program for metastasis initiation could be exploited to improve the diagnosis and treatment of CRC.