Potential Drivers of Acquired Resistance to Idelalisib in CLL Patients

Idelalisib is an isoform-specific PI3-kinase-δ inhibitor approved for the treatment of chronic lymphocytic leukemia (CLL). We seek to understand the biologic basis of the variability in depth of response among patients. We have previously reported that intrinsic resistance to idelalisib is associated with RAS/RAF/MAP2K1 mutations and constitutive ERK activation [Blood 2021; 138: 44]. Unlike these non-responder patients, some patients with initial response will acquire resistance to therapy over time, but molecular mechanisms of this acquired resistance to idelalisib remain elusive. To identify somatic mutations that fuel acquired resistance, we performed whole exome sequencing of serial PBMC samples from three CLL patients who responded to idelalisib, but later acquired resistance (average 4.1 yrs from start). We observed that all three patients acquired distinct somatic mutations during idelalisib treatment. Patient 1 harbored TP53 and BIRC3 at baseline, but at progression (5.3 yrs later) had acquired somatic mutations in RAF1, PDIA3, RB1, and ATM. Patient 2 had baseline mutations in SF3B1, BRCA2, and HIST1HIE but acquired additional mutations in PIK3R1 and IKZF3 at progression (4.4 yrs later). Patient 3 carried somatic mutations in TP53 and SF3B1 at baseline and acquired somatic mutations in DDX3X, NOTCH1, FBXW7, CD79B, NFATC1 and NFATC3 at progression (2.7 yrs later). To identify novel drivers of resistance and validate our findings from the initial three CLL patients, we sequenced progression samples from eight additional CLL patients following resistance to idelalisib treatment. In addition to patient 1, we detected mutations in BIRC3 in two additional CLL cases, and mutations in KRASand NRAS (both of which are known to aberrantly activate the MAPK pathway) in two and one CLL patients, respectively, separate from the cases with BIRC3 mutation. In addition to the PIK3R1 acquired mutation in patient 2, we also identified mutations in other PI3K pathway-related genes, such as PIK3CA, PIK3CG, andPLCG2. Since we only sequenced progression samples from these 8 patients, we cannot confirm whether the mutations were acquired or were present in the baseline samples. Overall, these results suggest that mutations in the RAS/RAF/MAP2K1 pathway, as well as in the PI3K pathway itself, and BIRC3 (NF-kB pathway), are associated with PI3K inhibitor resistance. To identify expression programs that may lead to idelalisib resistance, we performed comparative transcriptomic profiling of responding and resistant time point samples from the three initially studied CLL patients (patient 1, 2 and 3). In this analysis, we detected 214 differentially expressed genes with adjusted p-value < 0.05 and fold change of 1.5. GSEA analysis revealed enrichment of cancer related cell signaling pathways, most notably an enrichment for the MAPK pathway (FDR q-value- 0.04). We found that AICDA, LTK, and MAPK12 are overexpressed in resistant samples from the three original CLL patients with acquired resistance as compared to the matched responding time points. In a separate cohort of frontline patients treated with idelalisib, we also found that these same 3 genes were consistently overexpressed before, during and after treatment in early progressors after idelalisib discontinuation. Higher expression of AICDA in progression samples may drive resistance by inducing somatic mutations and/or altering the epigenome of the tumor cells in CLL patients. LTK receptor tyrosine kinase activates PI3K and MAPK pathways in hematopoietic cells. Previously we have reported that MAPK pathway activation plays a key role in primary resistance to idelalisib in CLL, hence overexpression of LTK is a possible mechanism of MAPK activation. We checked the phosphorylation/activation level of AKT and ERK1/2 at the responding and progression time points in the three original CLL patients with acquired resistance and observed that pERK levels are inhibited by idelalisib at baseline, but not at progression, in 2 patients (patient 1 and 2). In conclusion, we have identified potential drivers of acquired resistance to idelalisib in CLL patients, including MAPK pathway activation as in our prior study, and we continue with ongoing work evaluating the potential role of PI3K pathway mutations, BIRC3, AICDA and LTK in treatment resistance, since insights from these studies will help us guide therapy for CLL patients with refractory disease.