A Novel Spirocyclic Dimer (SpiD3) Resensitizes CLL Cells to Venetoclax

Chronic lymphocytic leukemia (CLL) is an incurable, heterogeneous B-cell malignancy with a dependence on B-cell receptor signaling and subsequent activation of nuclear factor-kappa B (NF-κB) for growth and survival. Almost all CLL cells evade apoptosis primarily through overexpressing the B-cell lymphoma/leukemia 2 (BCL2) protein, making it an attractive therapeutic target. Venetoclax (VEN), a BH3-mimetic that selectively inhibits BCL2, is highly cytotoxic to CLL cells and has shown high response rates in refractory/relapsed (R/R) CLL. Despite the clinical success of VEN in CLL, a subset of patients (~25%) fail to achieve minimal residual disease and succumb to progressive disease and/or drug resistance. Factors contributing to VEN resistance have been reported such as mutations in BCL2 (e.g., BCL2^G101V), marked NF-κB pathway activation, and increased expression of other anti-apoptotic BCL2 family proteins (e.g., MCL1, BCL-xL and BFL-1) that are not targeted by VEN. This indicates the need for novel therapeutic approaches for R/R CLL that bypass resistance mechanisms and/or target alternative B-cell survival pathways. We therefore hypothesize that targeting NF-κB signaling would augment the efficacy of VEN-based regimens and/or mitigate resistance to VEN in CLL. To investigate this, we used a novel spirocyclic dimer, SpiD3, that covalently binds to NF-κB proteins, IKKβ and RELA/P65 (Rana S, etal, 2022). We previously established the anti-leukemic properties of SpiD3, reporting the IC₅₀ below 1 μM in CLL cell lines (Eiken A, et al, 2021). To evaluate if SpiD3 can potentially enhance VEN cytotoxicity in CLL, we conducted synergy assays in CLL cell lines (HG3 and OSUCLL). Cell proliferation was measured using the MTS assay following a 72-hour treatment with increasing concentrations of SpiD3 and/or VEN. Consistent with our hypothesis, 0.5 μM SpiD3 showed strong synergy with 0.25 μM VEN in HG3 cells and with 0.125 μM VEN in OSUCLL , which is less than the reported plasma concentration of VEN in CLL patients (0.79 - 1.14 μM). Since, NF-κB activation is implicated in VEN-resistance, we next evaluated SpiD3 in a preclinical CLL model of acquired VEN resistance. We developed VEN resistance in OSUCLL cells through prolonged exposure to increasing concentrations of VEN until the cells remained viable in the presence of 22 μM VEN. Remarkably, SpiD3 elicited robust cytotoxicity in the VEN-resistant OSUCLL cells with an IC₅₀ of ~1 μM (Figure 1). Treatment of VEN-resistant OSUCLL cells with SpiD3 for 4 hours resulted in reduced expression of anti-apoptotic proteins (MCL1 and BFL-1) and increased levels of the pro-apoptotic protein, PUMA. This was coupled with marked PARP cleavage. In contrast to VEN, SpiD3 reduced P65 and MYC expression and attenuated phosphorylation of ERK (Thr202/Tyr204) and PRAS (Thr246), all factors known to contribute to VEN resistance. Further interrogation of differential gene expression following SpiD3 treatment in VEN-resistant cells is ongoing to delineate the molecular mechanism underlying SpiD3's anti-tumor properties in VEN-resistant CLL. In summary, our data (a) supports the efficacious combination of SpiD3 with VEN as a viable therapeutic approach in CLL to bypass NF-κB activation underlying VEN resistance and (b) introduces SpiD3 as an alternative therapeutic approach for VEN-resistant CLL patients.

Rana:University of Nebraska Medical Center: Patents & Royalties: "Dimers of covalent NFkB inhibitors” Natarajan, A. and Rana, S. PCT Int. Appl. (2018), WO 2018119177 A1 20180628. Natarajan:University of Nebraska Medical Center: Patents & Royalties: "Dimers of covalent NFkB inhibitors” Natarajan, A. and Rana, S. PCT Int. Appl. (2018), WO 2018119177 A1 20180628.