Combined Inhibition of BCR and IGF1R Signaling Leads to Synergistic Apoptosis Induction in Primary CLL Cells in Vitro
Authors: Schmidt-Salzmann Charlotte , Yaktapour Niuscha , Brummer Tilman , Zirlik Katja , Claus Rainer 
Published: October 2019

Abstract
Introduction: Chemoimmunotherapy has been the standard of care for chronic lymphocytic leukemia (CLL). However, novel therapeutic strategies targeting the B cell receptor (BCR) pathway like the BTK inhibitor ibrutinib and the PI3K delta inhibitor idelalisib have demonstrated superior results in clinical trials. Consequently, both substances have become standard in relapsed and refractory patients and have replaced conventional chemoimmunotherapy as treatment of choice in high-risk patients with deletion 17p or TP53 mutation. Despite their high clinical effectiveness, these drugs cannot achieve deep remissions as single agents and require continued treatment. Furthermore, first mechanisms of resistance such as BTK and PLCG mutations have been reported. The persistence of CLL under BCR signaling blockade could be explained by the activation of alternative pathways such as the insulin-like growth factor 1 receptor (IGF1R) pathway. In line, we have previously demonstrated IGF1R overexpression with concomitant IGF1R pathway activation in CLL patients and successful apoptosis induction by IGF1R inhibition in CLL cells. For solid tumors and multiple myeloma, the potential of IGF1R inhibition is currently being evaluated in clinical studies with the IGF1R inhibitor linsitinib. Here, we demonstrate synergistic effects on apoptosis induction by combined inhibition of BCR and IGF1R signaling.
Methods and Results: To evaluate a possible benefit from combined inhibition of BCR and IGF1R, we cultured primary CLL cells with previously determined subtoxic concentrations of ibrutinib (4µM), idelalisib (5 µM) and the IGF1R antagonist linsitinib (OSI-906, 4 µM) alone and in combination. Cell viability was analyzed using flow cytometry with Annexin/PI staining 48 hours and 96 hours after treatment. In vitro treatment of CLL patient samples (n=5) with single-agent ibrutinib, idelalisib and linsitinib as well as with the combination of ibrutinib and idelalisib caused a decreased viability of 90 ± 8% after 48 hours compared to the untreated controls. In contrast, the effect of combined inhibitor treatment with linsitinib and either ibrutinib or idelalisib led to a marked but not significant decrease in viability to 76 ± 6 %. In order to assess treatment efficacy in the context of microenvironment interaction, CLL cells (n=6) were co-cultured with the murine stromal cell line M2-10B4, and treatment started after 24 hours. Again, single agent treatment and combined ibrutinib/idelalisib treatment resulted in a comparable reduction of viability to 89 ± 7% for ibrutinib, 77 ± 5% for idelalisib and 77 ± 6% for ibrutinib/idelalisib. Combined BCR and IGF1R pathway inhibition using linsitinib with either ibrutinib or idelalisib led to a significant decrease in cell viability to 53 ± 11% and 45 ± 8%, respectively. This effect was considerably more pronounced than expected from the single-agent treatments. It exerted the combined BTK/PI3K delta inhibition significantly in all of the performed experiments and proved consistent after 96h. Notably, the strong synergistic effect of apoptosis induction through BCR and IGF1R inhibition could also be observed in specimens from two high risk CLL patients harboring deletion 17p. CLL cell viability was reduced to 38 ± 9% for linsitinib/ibrutinib and 30 ± 2% for linsitinib/idelalisib, respectively, compared to 65 ± 3% for the combination of ibrutinib and idelalisib.
Conclusion: In conclusion, we demonstrate that IGF1R inhibition constitutes a novel and effective therapeutic target for CLL therapy. We confirm previous data on effective apoptosis induction by IGF1R inhibition in CLL in vitro (Yaktapour N et al., Blood 2013;122:1621-1633) and demonstrate efficacy comparable to ibrutinib and idelalisib treatment. Combinations with the IGF1R inhibitor linsitinib show superior in vitro efficacy compared to single-agent treatment and to combined BTK/PI3K delta blockage. These results indicate synergistic effects of kinase inhibition, possibly due to blockage of (IGF1R) pathway recruitment. Current experiments are dissecting BCR and IGF1R pathway activity and testing IGF1R expression and phosphorylation as predictive biomarker. We therefore conclude that combined BCR/IGF1R inhibition is highly effective presumably leading to deep remissions, and we propose that translation of the in vitro data into clinical trials seems feasible.

Disclosures
No relevant conflicts of interest to declare.