Dual targeting of P13K and BCL-2 in ibrutinib-resistant MCL: A preclinical study

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma. Treatment of relapsed/refractory MCL with ibrutinib, a Bruton’s tyrosine kinase inhibitor (BTKi), is complicated by the yet unresolved challenge of BTKi resistance.¹ Primary resistance is displayed in around one-third of patients, with secondary resistance developing over time almost universally in the remainder.¹

In a preclinical study published in the Journal of Cellular and Molecular Medicine, Ye and Huang et al.¹ sought to target compensatory pathways that drive BTKi resistance. In ibrutinib-resistant (IR) MCL cells, high expression of BCL-2 and MCL-1 (both prosurvival/antiapoptotic proteins) is driven by sustained P13K-mTOR activation. They hypothesized that targeting both P13-kinase and BCL-2 signaling pathways would result in synergistic anti-MCL activity and overcome BTKi resistance.

Duvelisib is a dual P13Kδ/γ inhibitor that has shown preclinical activity in both ibrutinib-sensitive and IR MCL. Venetoclax, a BCL-2 inhibitor, has shown clinical efficacy in patients with MCL, particularly when in combination with ibrutinib. In chronic lymphocytic leukemia, duvelisib treatment increased sensitivity to venetoclax treatment and decreased BCL-2 expression.

Using in vitro and in vivo models of resistance, as well as post-ibrutinib treatment clinical samples, Ye and Huang et al. considered the efficacy of duvelisib and venetoclax combination in IR MCL cells. The study and its findings are summarized in this article.

Study design

Cell lines and patient samples:

• The following MCL cell lines were used: JeKo-1, Maver-1, Z138, JeKo-R, JeKo BTK KD cells, and JeKo-Luc cells.
• Human stroma cell line HS-5 cells were used as the tumor microenvironment (TME) attractant.
• Four IR MCL patient samples were obtained.

Duvelisib and venetoclax treatment on cell viability and signaling pathways:

• Four IR MCL cell lines (JeKo BTK KD, Jeko R, Maver-1, and Z138) and four patient samples were treated with increasing doses of duvelisib (0–12.5 µM), venetoclax (0–100 nM), and both drugs in combination at varying doses.
• Cell viability was detected at 24 hours and 72 hours in the patient samples and IR MCL cell lines, respectively, by annexin-V/PI staining.
• JeKo BTK KD cells were treated with 5 µM duvelisib and/or 100 nM venetoclax for 24 hours and harvested for reverse phase protein array (RPPA) analysis.
• Proteins with more than a twofold change between combination treatment with duvelisib and venetoclax and the vehicle control (solvent dimethylsulfoxide [DMSO]) were selected for heatmap generation.
• JeKo BTK KD and Z-138 followed the same treatment as above, with protein lysates collected for western blotting.

Cell migration in the tumor microenvironment (TME) and TME-mediated ibrutinib resistance:

• A transwell migration assay was performed using IR JeKo BTK KD cells and HS-5 cell monolayer as the TME attractant to investigate whether duvelisib and venetoclax in combination synergistically block directional cell migration.
• Ibrutinib-sensitive JeKo-1 cells were co-cultured with HS-5 cells to investigate the role of the TME in tumor phenotypic changes.
  • As with the primary IR models, cells were treated with increasing doses of duvelisib (0–12.5 µM), venetoclax (0–100 nM), and both drugs in combination at varying doses, and cell viability was assessed.

In vivo drug efficacy in mouse xenograft models:

• JeKo-Luc cells were injected subcutaneously into 6–8-week-old NOD SCID IL2Rγ null mice.
• Treatment began 3 days following inoculation with ibrutinib (50 mg/kg, oral, daily), venetoclax (50 mg/kg, oral, daily), and duvelisib (50 mg/kg, oral, daily) alone or in combination, or the vehicle alone for 3 weeks.
• Weekly tumor measurement was performed using an IVIS Spectrum In Vivo Imaging System.

Results

Cell viability and signaling pathways in response to duvelisib and venetoclax

All four IR MCL cell lines and three of the four patient samples (Patients 1,2, and 3) were only slightly responsive to duvelisib as a single agent.

• Two cell lines (Z138 and Maver-1) and two patient samples (Patient 1 and Patient 3) were sensitive to venetoclax as a single agent.
• A notable synergistic effect was observed in three of four cell lines and three of four patient samples, with a weaker combination effect observed in Maver-1 and Patient 4.
• The synergistic effect was confirmed by isobologram analysis.
• Cell apoptosis (30.3–97.0%) was induced by combination therapy (combination index, 0.36–0.77) in all tested cell lines.
• PI3K/AKT/mTOR, PKC, and PLK1 signaling pathways were predominantly downregulated in the combination-treated cells, with apoptosis-related proteins, such as caspases and histone-H3, the most upregulated.

Cell migration in the TME and TME-mediated ibrutinib resistance

• Cell migration towards the HS-5 monolayer was significantly reduced following a 30-minute pretreatment with venetoclax and duvelisib combination compared with controls (p = 0.027) and with either venetoclax or duvelisib alone (p = 0.036 and p = 0.056, respectively).

• Ibrutinib-sensitive JeKo-1 cells became IR when co-cultured with HS-5 cells in vitro (p < 0.0001).
• The same effect was observed in vivo, with the TME shifting the JeKo-1 phenotype from sensitive to IR with mice bearing JeKo-Luc-derived xenografts showing no response to ibrutinib treatment.
• When venetoclax and duvelisib were given in combination, this overcame the TME-associated acquired IR in the stroma/JeKo-1 co-culture (p = 0.0007). Similarly, JeKo-Luc-derived xenograft models were resistant to single-agent treatment, but sensitive to combination treatment (p = 0.003).

Conclusion

Combination treatment with duvelisib and venetoclax synergistically inhibited the cell growth of ibrutinib-resistant MCL models through downregulation of P13K/AKT/mTOR pathway and activation of pro-apoptotic signaling. Furthermore, the treatment combination inhibited TME-mediated cell migration and overcame acquired and TME-mediated IR in MCL cells both in vitro and in vivo. These promising results support dual targeting of P13K and BCL-2 as a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory MCL.