NF-κB Pathway Mutations Modulate Cell Survival and Ibrutinib Response In Chronic Lymphocytic Leukemia
Authors: Improgo Reina , Tiao Grace , Kiezun Adam , Wang Yaoyu , Werner Lillian , Sougnez Carrie , Tesar Bethany , Fernandes Stacey M , Vartanov Alexander R. , Hoang Kevin , Neuberg Donna S. , Getz Gad , Brown Jennifer R. 
Published: October 2019

Abstract
Constitutive activation of the NF-κB pathway is a critical feature of hematologic malignancies and is associated with increased lymphocyte proliferation and survival. Various mechanisms leading to altered NF-κB activation have been described in lymphomas, including activating mutations in and upregulation of NF-κB pathway genes. We have previously reported whole-exome sequencing results from a cohort of 160 CLL patients showing mutations in several NF-κB pathway genes (as defined in the Ingenuity Pathway Analysis database), including MYD88 (n=12), RIPK1 (n=4), NRAS (n=4), KRAS (n=3), CARD11 (n=1), IRAK4 (n=1), PIK3CA (n=1) and TRAF3 (n=1).
Here, we describe functional approaches to evaluate the biological significance of NF-κB pathway mutations. Using the CellTiter-Glo assay in serum-free conditions, we assessed the relationship between NF-κB pathway mutation status and survival of CLL cells in vitro. We found that CLL cells with no NF-κB mutations exhibited spontaneous apoptosis in vitro, with 3.3 - 39.5% viable cells remaining after 48 hours in culture (n=4 patient samples; n=5 replicates per patient). Similarly, cells harboring the MYD88 L265P mutation yielded 0.6 - 23.1% viable cells after 48 hours (n=5 patient samples; n=5 replicates per patient). In contrast, cells harboring RIPK1 Q375*, RIPK1 K559R, KRAS Q61H, CARD11 E756K, IRAK4 K400E, and PIK3CA I143V displayed apoptotic resistance, with 48.6 - 132.5% viable cells remaining after 48 hours (1 patient sample per mutation; n=5 replicates per patient). Similar findings were observed in the context of B-cell receptor activation via IgM stimulation, with IgM stimulation generally enhancing CLL cell viability (mean = 24% increase after 48 hours). In the case of RIPK1 Q375*, however, no further increase in viability was observed, suggesting that this truncating mutation may obviate the need for external survival signals.
To identify NF-κB mutations that might associate with susceptibility to different therapies, we examined the relationship between NF-κB pathway mutation status and sensitivity to the NF-κB inhibitor, SN50. Treatment with SN50 for 48 hours resulted in loss of viability in cells that were negative for NF-κB pathway mutations (43.3 - 98.9% decrease in viability with 5 μM SN50; n=4 patient samples; n=5 replicates per patient). Similarly, cells harboring the MYD88 L265P mutation responded to SN50 treatment (43.1 - 97.0% decrease in viability; n=5 patient samples; n=5 replicates per patient). In contrast, cells harboring RIPK1 Q375*, RIPK1 K559R, CARD11 E756K, and PIK3CA I143V were more resistant to SN50 treatment (18.6% increase - 38.9% decrease in viability; 1 patient sample per mutation; n=5 replicates per patient). These results suggest that specific NF-κB pathway mutations confer resistance to NF-κB inhibition.
The above results prompted the question of whether NF-κB pathway mutations may also confer resistance to the BTK inhibitor, ibrutinib, which has previously been shown to block NF-κB pathway activation by inhibiting the phosphorylation of p65 and preventing its nuclear translocation. To address this question, we treated cells with 2.5 μM ibrutinib for 48 hours. Ibrutinib treatment led to a loss in viability in cells that had no NF-κB pathway mutations (26.3 - 82.9% decrease in viability; n=4 patient samples; n=5 replicates per patient). Cells harboring MYD88 L265P mutations also appeared to be susceptible to ibrutinib (25.8 - 82.9% decrease in viability; n=5 patient samples; n=5 replicates per patient). Notably, cells harboring the RIPK1 Q375* and KRAS Q61H mutations appeared to be more resistant to treatment (7.0% decrease and 2.0% increase in viability, respectively; 1 patient sample per mutation; n=5 replicates per patient). Furthermore, RIPK1 Q375* and KRAS Q61H cells remained resistant to ibrutinib in the context of BCR activation. Though our study utilized a limited number of patient samples representing a variety of mutations, the results are suggestive that specific NF-κB pathway mutations are functional and may influence intrinsic CLL cell survival, responsiveness to IgM stimulation, and sensitivity to drug treatment. Identification of specific mutations that confer resistance to ibrutinib is of particular clinical interest for predicting response and understanding drug resistance.

Disclosures:
Brown: Novartis: Consultancy; Vertex: Consultancy; Sanofi Aventis: Consultancy; Onyx: Consultancy; Emergent: Consultancy; Celgene: Consultancy, Research Funding; Genentech: Consultancy; Pharmacyclics: Consultancy; Genzyme: Research Funding.