Blockade of PDGFRβ circumvents resistance to MEK-JAK inhibition via intratumoral CD8+ T-cells infiltration in triple-negative breast cancer

Abstract

Background Despite the increasing progress in targeted and immune based-directed therapies for other solid organ malignancies, currently there is no targeted therapy available for TNBCs. A number of mechanisms have been reported both in pre-clinical and clinical settings that involve inherent, acquired and adaptive resistance to small molecule inhibitors. Here, we demonstrated a novel resistance mechanism in TNBC cells mediated by PDGFRβ in response to JAK2 inhibition. Methods Multiple in vitro (subG1, western blotting, immunofluorescence, RT-PCR, Immunoprecipitation), in vivo and publically available datasets were used. Results We showed that TNBC cells exposed to MEK1/2-JAK2 inhibitors exhibit resistant colonies in anchorage-independent growth assays. Moreover, cells treated with various small molecule inhibitors including JAK2 promote PDGFRβ upregulation. Using publically available databases, we showed that patients expressing high PDGFRβ or its ligand PDGFB exhibit poor relapse-free survival upon chemotherapeutic treatment. Mechanistically we found that JAK2 expression controls steady state levels of PDGFRβ. Thus, co-blockade of PDGFRβ with JAK2 and MEK1/2 inhibitors completely eradicated resistant colonies in vitro. We found that triple-combined treatment had a significant impact on CD44 + /CD24 − stem-cell-like cells. Likewise, we found a significant tumor growth inhibition in vivo through intratumoral CD8 + T cells infiltration in a manner that is reversed by anti-CD8 antibody treatment. Conclusion These findings reveal a novel regulatory role of JAK2-mediated PDGFRβ proteolysis and provide an example of a PDGFRβ-mediated resistance mechanism upon specific target inhibition in TNBC. Electronic supplementary material The online version of this article (10.1186/s13046-019-1075-5) contains supplementary material, which is available to authorized users.

CX-5461 Enhances the Efficacy of APR-246 via Induction of DNA Damage and Replication Stress in Triple-Negative Breast Cancer

“…The breast cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium–4.5 g/L glucose, (Sigma Aldrich ® , Saint Louis, MO, USA) with 10% fetal bovine serum (FBS) as described previously [ 56 , 57 , 58 ].…”

'Off-the-shelf’ allogeneic antigen-specific adoptive T-cell therapy for the treatment of multiple EBV-associated malignancies

“…The Aqueous One Solution Reagent was added to each well (1:100 dilution in media) and the plate was incubated for 1 hour prior to assessing the optical density at 490 nm using a microplate reader. 33 …”

“…The Aqueous One Solution Reagent was added to each well (1:100 dilution in media) and the plate was incubated for 1 hour prior to assessing the optical density at 490 nm using a microplate reader. 33 Cytotoxicity assay Cytotoxicity assays were performed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega) according to the manufacturer's instructions. Assays included three biological replicates per EBV-associated cancer cell line, in triplicate.…”

Cep55 overexpression promotes genomic instability and tumorigenesis in mice

“…The following are additional antibodies used in this study: Cell Signaling antibodies: PARP (#9542), pAKT S473 (#4060), AKT (#9272), pPdk1 S241 (#3061), Pdk1(#3062) Chk1 (2G1D5) (#2360), p-GSK-3β (Ser9) (#9336), GSK-3β (#9315), p-Histone H3 (#9706) (1:1000 dilution); Millipore antibody: Chk2 (1:500 dilution) (Clone 7) (05-649), γ-H2ax (1:1000 dilution) S139 (05-636); BD Pharmingen antibody: β-actin (1:2000 dilution) (612656); Bethyl antibody: pKap1 (S824) (1:1000 dilution) (A300-767A). Immunoprecipitation was performed as per our previous publication 45 . Immunodetections were performed using Bubr1 (ab4637), Mad2 (CST4636S) and CDC20 (CST14866A).…”