Diminished Proteolytic Shedding of Receptor Tyrosine Kinases Mediates MEK Inhibitor Resistance in Triple-Negative Breast Cancer

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Direct sheddase inhibition leads to AXL accumulation, compensatory MAPK signaling, and enhanced proliferation. a) Change in total- and phospho-RTK levels following 24 hr 8894 treatment; each dot represents one of 19 cell lines tested (see Sl-2a for data). b) Change in the protein levels listed on the left were measured across a panel of TN8C cell lines following 24 hr 8894 treatment (see Sl-2b for data). The heat-map shows hierarchical bi-clustering of the spearman correlations between protein drug responses, across the panel of cell lines. Co-clustering with surface-AXL is highlighted in green. c) 8894 increases phosphosignaling in an R428-dependent manner, measured by bead-based immunoassay in MDAM8231 (n=B reps). d) AXL siRNA reduces compensatory p-Jnk signaling, measured following 3 hr drug treatment and 48 hr siRNA treatment in MDAM8231 (*p=0.014, pooled two-sided t-test, n=2 reps I cond). e) siRNA and proA10 inhibitor reduce 24 hr supernatant accumulation of AXL compared to their respective controls, measured by ELISA (*p<0.05; two-tailed student's t-test; n􀀚2 reps). f) Live-cell immunostaining shows A10 and A17 siRNA increase surface AXL in MDAM8231 and MDAM8157, 72 hr post-treatment. g) 8894 increases mitotic index, measured by FACS, in an R428-dependent manner (bars denote p<0.05; n􀀚4 total reps; pooled two-tailed student's t­test). h) A17 siRNA increases cell count only in the absence of the AXL inhibitors R428 and MP470  (*p=0.047; two-tailed student's t-test; n=4 reps I cond.), measured 24 hr after AX.Li, 72 hr after siRNA treatment. MEK inhibition reduces sheddase activity via increased homo-dimerization and association with TIMP1. a) MEKi reduces A10 and A17 catalytic activities in MDAMB231, inferred using PrAMA (*p<0.05; two-tailed student's t-test; n=4 reps I cond.). b) Phospho-A17 decreases following MEKi (p=0.03, pooled two-tailed student's t-test; n=2 reps I cond; see Sl-3c for western blots). c) A17 homodimerization increases with PD325901 treatment in MDAMB231, MDAMB157, and BT549  (p=0.001; pooled two-tailed student's t-test; n=6 total reps; see Sl-3d for blots). d) 24 hr PD325901 treatment decreases supernatant TIMP-1 and -3 in MDAMB231, measured by Ab-microarray (*p<0.05; two-tailed student's t-test; n=4 reps). e) Live-cell immunostaining shows TIMP1 siRNA reduces surface AXL in MDAMB231, 24 hr after PD325901 treatment and 72 hr after siRNA treatment (*p<0.05; two­tailed student's t-test; n=3 reps). f) Live-cell immunostaining shows MEKi increases surface TIMP1 in MDAMB231 and MDAMB157 (bar denotes p<0.05; pooled two-tailed student's t-test; n􀀿2 reps I cond). g) Live-cell immunostaining shows surface TIMP1 decreases, 72 hr after A10 and A17 siRNA treatment in MDAMB231 (*p<0.05; two-tailed student's t-test; n􀁊2 reps I cond). Plasma from melanoma patients was assayed for soluble RTK levels (including AXL and MET) before treatment with dual BRAFi/MEKi therapy; Kaplan-Meier analysis based on average levels of 7 RTKs shows significant correlation with disease outcome (see Miller et al., Cancer Discov. 2016 Apr;6(4):382-99).
Categories
Inventors
Professor Douglas Lauffenburger
Department of Biological Engineering, MIT
External Link (web.mit.edu)
Professor Linda Griffith
Department of Biological Engineering, MIT
External Link (lgglab.mit.edu)
Professor Frank Gertler
Department of Biology, MIT
External Link (web.mit.edu)
Aaron Meyer
Koch Institute for Integrative Cancer Research, MIT
External Link (asmlab.org)
Miles Miller
Department of Biological Engineering, MIT
Madeleine Oudin
Koch Institute for Integrative Cancer Research, MIT
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

Methods of Reducing Kinase Inhibitor Resistance

US Patent Pending 2016-0067250
Publications
Reduced Proteolytic Shedding of Receptor Tyrosine Kinases Is a Post-Translational Mechanism of Kinase Inhibitor Resistance
Cancer Discovery, April 1, 2016

Applications

This invention diagnoses kinase inhibitor resistant cancer and also decreases resistance to kinase inhibitors for improved cancer treatments.

Problem Addressed

Molecular-targeted cancer therapeutics eventually fail due to cancer’s ability to become resistant to drugs. How carcinogenic cells achieve this resistance is poorly understood, with genetic and gene expression changes accounting for only a small percentage of their adaptability. This invention is a method to reduce cancer’s resistance to anti-cancer therapeutics, and may be used for triple-negative breast cancer (TNBC), melanoma, ovarian cancer, and other indications where MAPK pathway activation is prominent. The invention may also be used to aid in selecting patients for targeted combination therapies and in monitoring patients for treatment response and mechanisms of drug resistance.

Technology

This technology is based on the discovery that decreased proteolytic shedding of surface receptors, which provide negative feedback on signaling network activity thereby driving post-translational “bypass” signaling pathways, can be caused by anti-cancer kinase inhibitor treatments leading to resistance to those very same therapeutics. Inhibition of multiple signaling pathways, in particular MAPK signaling through MEK1/2, drives “bypass” signaling by causing diminished A Disintegrin and Metalloproteinase (ADAM)-mediated receptor tyrosine kinase (RTK) shedding including MET, HER2, HER4, and especially AXL RTKs. Targeted MEK inhibition (MEKi) has been identified as a promising therapeutic strategy since RAF/MEK /ERK mitogenic phospho-signaling is up-regulated in many cancers including TNBC.  MEK, BRAF, p38, JNK and PI3K kinase inhibition have been shown to reduce RTK shedding and hence gives rise to the bypass signaling. This technology increases sensitivity of cancers under treatment with kinase inhibitors by blocking the bypass signaling pathways of RTKs and/or by encouraging RTK receptor shedding.

Advantages

  • May be used in conjunction with kinase inhibitor cancer treatments to decrease resistance
  • May be used to identify patients likely to respond to kinase inhibitor treatments and combinations thereof