High-Level ROR1 and BCL2, Cancer-Stemness, and BCL2 Mutations Associate with Venetoclax Resistance in Chronic Lymphocytic Leukemia

Venetoclax (Ven) is an inhibitor of BCL2 that is highly active in patients (pts) with chronic lymphocytic leukemia (CLL), effecting remissions without detectable minimal residual disease (MRD), particularly when used in combination with an anti-CD20 mAb (Seymour et al., N Engl J Med, 2018). However, pts can have persistent detectable MRD (i.e. ≥10^-4 CLL cells by flow cytometry) after ≥1 year (yr) of Ven therapy (V-Rx); such pts are at risk for developing progressive disease (PD) even with continued V-Rx (Kater et al., J Clin Oncol, 2019). Evaluation of CLL cells from such pts may define biologic markers for pts who are likely to have persistent MRD after 1 yr of V-Rx and elucidate potential mechanism(s) of Ven resistance. We examined the CLL cells of pts (N=13) who had persistent MRD after ≥1 yr of V-Rx; 8 developed PD after a median time of 2 yrs on V-Rx and were designated as being in subgroup A1. Pts who had persistent MRD without PD after ≥1 yr of V-Rx were designated as being in subgroup A2 (N=5). For comparison we examined the pre-treatment (pre-Rx) CLL cells of pts who cleared MRD within 1 yr of therapy (N=5), and designated such pts as being in group B. The CLL cells of most pts expressed unmutated IGHV (i.e. 7/8 pts in A1, 3/5 pts in A2, and 5/5 pts in B). However, a high proportion of the pts with MRD after ≥1 yr of V-Rx had pre-Rx CLL cells with a complex karyotype and del17p (i.e. 5/8 pts in A1 and 2/5 pts in A2); whereas none of pre-Rx CLL cells of group B had a complex karyotype or del17p. We examined CLL cells for intracellular BCL2 and surface ROR1, which prior studies showed were correlative in CLL (Rassenti et al., Proc Natl Acad Sci U S A, 2017). The pre-Rx CLL cells of pts from subgroups A1 and A2 expressed significantly higher levels of ROR1 and BCL2 than the pre-Rx CLL cells of group B (P=0.03 and 0.0002, respectively, Mann-Whitney test). Furthermore, the CLL cells of pts with PD on V-Rx expressed significantly higher levels of ROR1 and BCL2 than the already high-levels expressed by the pre-Rx CLL cells of these same pts (P=0.002 and 0.01, respectively, Paired t test). We did not observe temporal changes in ROR1 or BCL2 in serial CLL samples collected over a comparable time interval from a comparator group of pts with adverse cytogenetics who did not receive V-Rx. We performed RNA sequencing with a mean of 70-million reads per sample on negatively-selected pre-Rx CLL cells from each pt, and on the isolated CLL cells from each of 6 pts in subgroup A1 when they had PD on V-Rx. Transcriptome analyses revealed the cancer-stemness gene-expression signature influenced by ROR1-signaling and associated with poorly-differentiated cancers (Choi et al., Cell Stem Cell, 2018; Malta et al., Cell, 2018) was significantly enriched in pre-Rx CLL of pts in subgroups A1 and/or A2 compared to group B (A1 and/or A2 vs. B had FDR q values of <0.001). We also found the transcriptomes of CLL cells from pts with PD on V-Rx had a significantly greater enrichment in the cancer-stemness gene-expression signature than that of the pre-Rx CLL cells of the same pts (FDR q value <0.001)! We identified the BCL2^G101V mutation found earlier (Blombery et al., Cancer Discov, 2019) in the CLL cells of 3 of 6 pts with PD in subgroup A1 at allelic frequencies of less than 20%; this BCL2^G101V mutation was not detected in pre-Rx CLL samples. We identified a new nonsynonymous BCL2 mutation at an allelic frequency of 49.3% in the CLL cells of 1 pt with PD who lacked the BCL2^G101V mutation; this pt's pre-Rx CLL cells did not harbor detectable levels of this BCL2 mutation, which we deduce alters the BCL2 BH3-binding pocket. In summary, this study reveals that pts with CLL cells having complex cytogenetics, del17p, high-level expression of ROR1 and BCL2, and/or transcriptomes enriched for cancer-stemness may be at greater risk for having persistent MRD at ≥1 yr of V-Rx. Furthermore, the CLL cells of pts who develop PD on V-Rx have significantly higher levels of ROR1 and BCL2, BCL2 mutations, and transcriptomes with greater enrichment of the cancer-stemness signature than that of CLL cells from the same pts prior to V-Rx, implying that CLL cells resistant to Ven have greater cancer-cell de-differentiation. Because of the high frequency of mutations in BCL2 for pts with PD on V-Rx, strategies targeting ROR1 (Choi et al., Cell Stem Cell, 2018), rather than higher doses of Ven, may be more effective in mitigating the risk of PD in high-risk pts treated with Ven-based regimens.