Felipe Samaniego

Background: Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and approximately 30% of the patients develop relapsed/refractory disease that becomes a major cause of mortality and morbidity. Several reports indicated that the BTK inhibitor ibrutinib successfully blocks B-cell receptor signaling and shows clinical benefit in leukemia and lymphomas, including mantle cell lymphoma [23422267] and DLBCL [26193343], for which ibrutinib is FDA-approved. In phase I/II clinical trials, ibrutinib elicited an overall response rate of 68% in patients with relapsed/refractory MCL. However, in spite of these encouraging results, responses are variable and generally incomplete, acquired resistance is common, and recurrence is anticipated [26430726]. We undertook a study of factors underlying acquired ibrutinib resistance (IR) in initially ibrutinib-sensitive DLBCL cell lines.

Methods: IR DLBCL cell lines were generated by continuous culture of parental (PT) cell lines in increasing concentrations of ibrutinib, up to a maximum concentration of 10µM. Once established, IR cell lines were removed from ibrutinib, expanded, and cultured under the same conditions as the PT cell lines for further experiments. Gene expression profiling (GEP) of IR and PT populations was performed on Agilent 4 x 44K gene microarrays.

Results: Of five ABC DLBCL cell lines tested, two (OCI-LY3, U2932) were initially resistant to ibrutinib (IC₅₀>10µM).Three (TMD8, OCI-LY10, HBL1) were initially sensitive (IC₅₀ < 10 nM), but chronic exposure to ibrutinib generated syngeneic versions with IC₅₀ > 5µM. In comparison to PT versions of these cell lines, IR cells did not form clumps in suspension cultures, displayed irregular cell morphology, elevated colony formation ability in methylcellulose matrix, and had higher proliferation rate. Western blots and GEP data showed increased expression by IR cell lines of IAP family members survivin, cIAP2, and oncogenic BCL2 and BCL6. Reduced B-cell receptor signaling, and enhanced PI3K-Akt activity was identified in IR cell lines. Analysis of PI3K isoforms revealed up-regulation of PI3Kα and PI3Kβ with decreased expression of PI3Kδ and PTEN (PI3K negative regulator). Given the enhanced PI3K isoform expression with IR, we treated cell lines with KA2237, a PI3Kβ/δ isoform targeting drug, and observed reduced metabolic activity (survival) of IR cells compared to PT cell lines.

Conclusion: This study highlights that changes in a regulator (PTEN) and mediator (p110β) of PI3K/AKT signaling have important roles in the development of ibrutinib resistance in DLBCL. Treatment with KA2237 may provide a better outcome for ibrutinib-resistant DLBCL.