MAPK-ERK Pathway Activation: The Achilles' Heel of IL-7R‒Induced Steroid Resistance

INTRODUCTION. Mutations in the IL-7R signaling pathway (e.g.IL7Ra, JAK1/3, PTPN11, NF1, STAT5B, N/KRAS or AKT) are associated with steroid resistance and inferior relapse-free survival. These mutant molecules drive strong activation of the JAK-STAT, MAPK-ERK and/or PI3K-AKT signaling pathways. Also, activation of wild type RAS or AKT-reflecting physiological IL7-induced signaling-drives steroid resistance. These observations extend the findings in preB-ALL where RAS mutations were related to steroid resistance. Impaired transcription of pro-apoptotic BIM-an essential direct transcriptional target gene of the glucocorticoid receptor NR3C1 that mediates steroid-induced apoptosis-is observed in various steroid resistant PDX models.

AIM. Given the central role of BIM in steroid-induced apoptosis, we investigated how mutations in IL7R or downstream signaling components would affect the function of BIM and other BCL2 protein family members (BCL2, BCLXL and MCL1) in steroid-exposed T-ALL.

RESULTS. We generated doxycycline-inducible SUP-T1 and P12 Ichikawa cell line models that can overexpress specific IL-7R signaling mutants. Expression of mutant JAK1, NRAS or IL7Ra molecules results in robust activation of the MAPK-ERK pathway that confers steroid resistance. As a target of NR3C1, BIM transcription was not impaired in these steroid treated cell lines. Therefore, we investigated post-transcriptional mechanisms of steroid resistance. We observed strong phosphorylation of BIM downstream of MAPK-ERK activation under steroid treated and untreated conditions. Exposure to each of three MEK-inhibitors CI1040, Selumetinib or Trametinib abolished not only phospho-ERK but also phosphorylation of BIM. To study the consequences of BIM phosphorylation in relation to binding and inactivation of anti-apoptotic proteins, we immunoprecipitated BIM. Increased BIM levels following steroid exposure was related to increased binding to BCL2, BCLXL and MCL1 in steroid-sensitive control cells. In contrast, activation of mutant JAK1 and NRAS molecules resulted in phosphorylated BIM and reduced binding of BIM to these anti-apoptotic BCL2 family members. This indicates that preventing ERK-mediated phosphorylation of BIM enhances steroid responsiveness. T-ALL patients with activating IL-7R signaling mutations may therefore benefit from treatment combining steroids and MEK inhibitors. Indeed we observed strong synergy between MEK inhibitors and steroids in primary T-ALL patient samples.

CONCLUSIONS AND FUTURE PERSPECTIVES. Treatment with MEK-inhibitors Selumetinib or Trametinib was shown effective in reversing steroid resistance, and therefore strongly synergize with steroid treatment. Our data highlights the importance of the dynamic interplay between BIM and anti-apoptotic BCL2 family members in MAPK-ERK-driven steroid resistance. Therefore, we are exploring whether MEK inhibitors (that prevent phosphorylation and inactivation of BIM) combined with BH3-mimetics (that block anti-apoptotic family members) will further enhance restoration of steroid responsiveness in IL-7R signaling mutant T-ALL patients. As physiological IL7-induced signaling is sufficient to raise cellular steroid resistance, these inhibitors could be applied for all IL-7R signaling-dependent T-ALL patients, also in the absence of IL-7R signaling mutations. Refractory/relapse T-ALL patients are now eligible for the current phase 1/2 MEK-inhibitor‒dexamethasone (SeluDex) trial. We are using mass-spectrometry to determine the exact phospho-sites in BIM that drives steroid resistance that can serve as a valuable biomarker.