1034 - Venetoclax resistance induced by activated T cells can be counteracted by sphingosine kinase inhibitors in chronic lymphocytic leukemia

Introduction:
The treatment of CLL patients with venetoclax-based regimens has demonstrated efficacy and a safety profile (Roberts et al. 2016; Mato et al. 2018), but the emergence of resistant cells and disease progression is a current complication (Leverson and Cojocari 2018; Jayappa et al. 2021). We previously reported that the sphingosine kinase 1 and 2 (SPHK1/2) inhibitor, SKI-II enhanced the in vitro cell death triggered by fludarabine, bendamustine or ibrutinib and reduced the activation and proliferation of CLL cells (Almejún et al. 2017). Since we previously showed that autologous activated T cells (aaT) from CLL patients favor the generation of venetoclax resistance (Elias et al. 2022;Elías et al. 2018), we aimed to determine whether SPHK inhibitors could overcome this negative (undesired) effect.

Methods:
Peripheral blood mononuclear cells (PBMC) were obtained from twenty-eight unrelated CLL patients that were free from clinically relevant infectious complications and were treatment naïve or without treatment for ≥3 months before the investigation began. To evaluate the effect of SPHK inhibitors on the generation of venetoclax resistance, PBMC were cultured with anti-CD3 antibody (aCD3) in order to induce T cell activation, with or without SKI-II (SPHK1/2 inhibitor) or opaganib (SPHK2 inhibitor) for 72h, in the presence of DMSO (vehicle) or clinically relevant doses of venetoclax (ven, 0,2µM) during the last 24h of culture. CLL cell survival was evaluated by flow cytometry and these values were employed to calculate venetoclax resistance index (VRI) for each patient as follows: VRI = (aCD3+Ven/aCD3) * (control/Ven). CLL cell proliferation (Ki-67) and activation (CD86, PD-1 and PDL-1) and T cell activation (CD40L) were evaluated by flow cytometry. SPHK1/2, BCL-XL and MCL-1 expression were evaluated by western blot in viable leukemic cells purified with magnetic beads. To assess the effect of SPHK inhibitors on already venetoclax-resistant cells, we first generated resistant cells, which were then cultured with SPHK inhibitors and venetoclax.

Results:
We found that both SPHK inhibitors reduced the upregulation of CD86, PD-1, PDL-1 (Figure 1A) and Ki-67 (Figure 1B) on CLL cells activated by aaT cells. SPHK inhibitors reduced the expression of CD40L induced on CD4+ cells upon CD3 crosslinking (Figure 1C) without affecting their survival (Figure 1D). More importantly, SKI-II and opaganib prevented the generation of venetoclax-resistance induced by aaT cells (Figure 1E) by reducing the upregulation of BCL-XL and/or MCL-1 on malignant cells (Figure 1F).
In addition, the presence of aaT cells enhanced SPHK2 expression on CLL cells, which was higher in those that survived to venetoclax (Figure 2A), while SPHK1 expression was not consistently modified (Figure 2B). Moreover, when the public data set RNA-Seq GSE192685 was analyzed, we found that 5 out of 7 CLL samples showed higher SPHK2 expression at progression on venetoclax therapy compared to prior-treatment (Figure 2C).
Finally, to determine whether SPHK inhibitors can affect the survival of already venetoclax-resistant cells, PBMC from CLL patients were cultured with or without aCD3 for 72h, with DMSO or venetoclax during the last 24h. After that, cells were washed and cultured again with DMSO, SKI-II or opaganib for another 96h, combined with DMSO or venetoclax during the last 24h of culture (Figure 2D). Because most of CLL cells die in control cultures with venetoclax (Figure 2E), this condition was not assessed. As expected, CLL cells that came from control cultures rapidly died in response to venetoclax and were not affected by SPHK inhibitors (Figure 2F). In CLL cells that came from aCD3+DMSO cultures, as previously, SPHK inhibitors promoted venetoclax-induced cell death (Figure 2G). Of note, venetoclax-resistant CLL cells that came from aCD3+venetoclax cultures died in response to opaganib alone (Figure 2H). Moreover, even though 64% of already venetoclax-resistant cells survive to a second drug exposure, 50% of these cells died due to the presence of venetoclax in combination with SKI-II and 75% in combination with opaganib (Figure 2H, right columns).

Conclusion: Venetoclax-resistant CLL cells express high levels of SPHK2. Both SPHK inhibitors reduce the activation and proliferation of CLL cells, diminish the generation of venetoclax-resistance and re-sensitize already venetoclax-resistant CLL cells to the drug, suggesting that the inhibition of SHPK2 may be involved in this process. Our results highlight the therapeutic potential of SPHK inhibitors in combination with venetoclax as a promising treatment option for the patients.