Key Points We provide a functional analysis of IGF1R expression in primary human B-CLL. Sorafenib reduces IGF1R expression in B-CLL.

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Chronic lymphocytic leukemia (CLL) remains incurable with current state of the art therapy creating the need for novel therapeutic concepts. Kinase inhibitors represent a promising strategy in the treatment of various malignancies including CLL. However, based on the recent experience with other targeted therapy compounds used as single agents, it appears important to identify additional targets and to evaluate therapeutic combinations targeting two or more critical signaling hubs in CLL cells. This strategy is likely to counteract the development of drug resistance more efficiently. We and others recently showed that the Raf/MEK/ERK pathway plays a critical role for the in vitro survival of CLL cells and demonstrated that drugs such as sorafenib targeting all Raf-isoforms and other kinases, induce apoptosis. Here, we provide a detailed analysis of various B-Raf inhibitors, including sorafenib, PLX4720 (vemurafenib tool compound) and dabrafenib on apoptotic pathways in primary human CLL cells and their impact on CLL cell survival alone or in combination with MEK (U0126), dual phosphoinositide-3 kinase/mTOR inhibitor (BEZ235), and IGF1R inhibitors (AG1024, Picropodophyllin (PPP)). 10µM sorafenib, a concentration comparable to plasma levels of this drug in patients, strongly induced apoptosis in CLL cells, while the more specific B-Raf inhibitor PLX4720 did not affect viability. This discrepant finding may be attributed to PLX4720’s well-described paradoxical ERK activation reported in various solid tumor types lacking BRAF mutations and containing increased Ras-GTP levels. Indeed, only sorafenib reduced ERK phosphorylation in CLL cells, while PLX4720 treatment even enhanced ERK activation. Interestingly, not only the B-Raf inhibitors PLX4720 or dabrafenib, but also lower doses (1-5µM) of sorafenib induced paradoxical ERK activation. Most likely, at these concentrations drug-bound Raf molecules act as potent allosteric activators on drug-free Raf monomers, thereby leading to an increase in ERK pathway activity. Importantly, paradoxical ERK activation coincided with enhanced viability. In line with findings in melanoma cells, BRAF (V600E) mutated CLL cells showed no paradoxical ERK activation and were effectively killed by the respective compounds. To identify inhibitor combinations reducing the in vitro survival of BRAF wild-type CLL isolates, we combined the B-Raf inhibitors with the MEK inhibitor U0126 or the dual PI3K/mTOR inhibitor BEZ235 observing reduced ERK phosphorylation and significantly enhanced rates of cell death (p<0.0009 for U0126; p<0.01 for BEZ235). This indicates that the additional MEK or PI3K inhibition counteracts paradoxical ERK activation in vitro and may overcome the resistance to apoptosis induction mediated by paradoxical ERK activation. Likewise, we tested combinatory effects of IGF1R inhibitors (AG1024, PPP) with suboptimal sorafenib doses and observed a significantly enhanced cell death for the combinations AG1024 and sorafenib (p<0,0001) as well as PPP and sorafenib (p<0,0001). In conclusion, this is the first description of a paradoxical ERK activation by Raf inhibitors in CLL cells with unmutated BRAF. Our observation of paradoxical ERK activation after treatment with suboptimal sorafenib doses may be of clinical importance since sorafenib is currently investigated in clinical trials in several malignancies including CLL. Our findings imply that Sorafenib plasma concentrations should be monitored when used for the treatment of CLL since lower plasma levels might promote paradoxical ERK activation and CLL progression. Furthermore, our results show the potential of therapies combining kinase inhibitors: additional MEK or PI3K inhibition or the concomitant inhibition of both pathways by IGF1R inhibitors may overcome paradoxical ERK activation. Furthermore, given our recent observation that the IGF1R is overexpressed in CLL cells, its inhibition by IGF1R kinase inhibitors might be advantageous over the single inhibition of MAPK or PI3K pathway components. Disclosures: No relevant conflicts of interest to declare.

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.

3864 Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B lymphocytes. For decades, nucleoside analogs, alkylating agents, and immunotherapeutics have remained the mainstay in treating this disease. Despite major advances in this field, CLL remains incurable with standard therapy. In recent years, preclinical and early clinical data on the use of kinase inhibitors have sparked new hope in the treatment of CLL. The multikinase inhibitor sorafenib, targeting RAF, platelet-derived growth factor receptor (PDGFR), KIT, FMS-like tyrosine kinase 3 (FLT3), and vascular endothelial growth factor receptor (VEGFR), has been approved for the treatment of renal cell carcinoma and hepatocellular carcinoma. Recent studies suggested that CLL cells might be also susceptible to this compound, however the precise mode of action in CLL cells remains elusive. In this study, we identified the Insulin-like growth factor receptor-I (IGF1R) pathway as novel target of sorafenib inducing cell death in CLL cells. Treatment with 10 μM of sorafenib significantly increased apoptosis in primary CLL cells as determined by AnnexinV/PI staining via flow cytometry. Commensurate with its RAF inhibiting properties, the apoptotic effect of sorafenib was accompanied with ERK pathway inhibition. Moreover, sorafenib treatment decreased phosphorylation of SRC and AKT, molecules implicated with IGF1R and insulin receptor (IR) signaling. Interestingly, the latter were strongly expressed in primary CLL cells compared with healthy B cells. Similar to sorafenib, 24 hour treatment of CLL cells with the three structurally distinct IGF1R inhibitors Picropodophyllin, AG1024, and Linsitinib significantly increased apoptosis compared with vehicle control resulting in decreased phophorylation of MEK, ERK, SRC, and AKT. Sorafenib and the IGF1R inhibitor AG1024 also downregulated the expression of IGF1R on CLL cells but not on healthy B cells. To test whether sorafenib modulates IGF-1 binding and thereby influences the IGF1R activation, we biotinylated recombinant IGF-1 and tested its binding to the IGF1R via flow cytometry. We observed a reduced IGF-1 binding after sorafenib treatment. IGF-1 binding after treatment with different IGF1R inhibitors was performed as an internal control. In order to further establish the functional relevance of IGF1R expression in CLL, we performed IGF1R specific and non-silencing siRNA experiments in primary CLL cells. In line with our previous results, IGF1R knockdown resulted in a significant decrease of cell viability and in downregulation of RAF-1 expression, and MEK, ERK, SRC, and AKT phosphorylation. The stromal microenvironment protects CLL cells from spontaneous and drug-induced apoptosis. Sorafenib, AG1024, and Picropodophyllin counteracted the protective effect of microenvironmental factors simulated by the presence of the murine stromal cell line M210B4, the chemokine CXCL12, and the integrin CD49d. Finally, we used the Eμ-Tcl1 transgenic mouse model to validate these results in vivo. Male and female mice (n=8) were treated with 25 mg/kg of the IGF1R inhibitor Linsitinib per oral gavage for 7 days and the amount of CD5/CD19 positive cells was determined flow cytometrically at different time points. We observed a reduction of CD5/CD19-positive cells by 26,1% and 23,2% after 4 and 8 days of treatment, respectively. Our results provide a novel mechanism of action of the multikinase inhibitor sofarenib in CLL cells by blocking IGF1R mediated signaling. IGF1R inhibition by itself induced apoptosis in CLL cells in vitro and in vivo, thus identifying IGF1R as promising target for therapeutic approaches and proposing IGF1R inhibitors for clinical assessment in the therapy of CLL. Disclosures: No relevant conflicts of interest to declare.