Impaired rRNA Synthesis Contributes to BCL-2 Induced Chemoresistance in Diffuse Large B-Cell Lymphoma

Overexpression of the antiapoptotic oncogene BCL-2 predicts poor prognosis in Diffuse Large B-cell Lymphoma (DLBCL) treated with anthracycline-based chemoimmunotherapy. Anthracyclines exert their anticancer activity by multiple mechanisms, however, a major contribution to their cytostatic and cytotoxic effects involves the inhibition of ribosome biogenesis (RiBi) through rRNA transcription blockade. RiBi inhibitors induce p53 stabilization through the ribosomal proteins (RPs)-MDM2-p53 pathway, the levels of stabilized p53 being dependent on baseline rRNA synthesis rate.

Despite these notions, the correlation between the baseline RiBi rate, BCL-2 levels and DLBCL outcome following chemoimmunotherapy treatment has not been investigated.

In this study we first confirmed that the standard CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy induces a potent ribosome biogenesis inhibition in DLBCL cell lines by hindering rRNA synthesis, as assessed by 45s rRNA qPCR. This activity was mainly attributable to doxorubicin, which mirrored the effects of well-known RiBi inhibitors (actinomycin D and CX-5461) on rRNA transcription. According with the p53-dependent mechanism of action of RiBi inhibitors, pharmacologic inhibition of rRNA synthesis did not translate in significant cytotoxicity in TP53 mutant DLBCL cell lines. Among TP53 wt cell lines, maximal efficacy was observed in the BCL-2 negative SUDHL-5 cell line, with BCL-2 positive TP53 wt cell lines being less sensitive. Enforced BCL-2 overexpression with a TET-ON inducible system rendered SUDHL-5 cells resistant to CHOP and single agent RiBi inhibitors. Importantly, the BH3-mimetic venetoclax could not fully reverse BCL-2 induced resistance. Following these observations, we hypothesized that BCL-2 could modulate RiBi inhibitors activity with BH3-independent mechanisms.

Enforced BCL-2 expression determined a reduced 45s rRNA synthesis rate in DLBCL cells, which resulted in a modest increase in the fraction of cells in the G1 phase of the cell cycle, in the absence of apoptosis.

In line with the known direct relationship between baseline RiBi rate and the amount of stabilized p53 upon rRNA synthesis blockade, BCL-2-associated reduction of baseline rRNA synthesis resulted in attenuated p53 induction after treatment with RiBi inhibitors, as determined by western blot assay. RNA-seq experiments performed in SUDHL-5 cells treated with RiBi inhibitors in the presence or absence of BCL-2, indicated that enforced BCL-2 overexpression attenuated the induction of p53 target genes. In line with this, combinatory strategies aimed at boosting p53 activation, proved to be effective in vitro in BCL-2 overexpressing DLBCL cells and in vivo in BCL-2 positive patient-derived xenograft models, synergizing with venetoclax. In fact, combinations of RiBi inhibitors with drugs stabilizing p53 in a RiBi-independent manner, such as MDM2i or etoposide, restored efficient p53 stabilization in BCL-2 overexpressing DLBCL models.

We next investigated whether differences in baseline RiBi rate may influence the outcome of DLBCL patients. For this goal, tissues from 2 independent DLBCL cohorts (of 83 and 46 patients respectively) treated with first-line anthracycline-based chemoimmunotherapy, were profiled with silver staining of nucleolar structures (AgNOR staining) to determine the nucleolar area (indicative of ribosome biogenesis rate), and with a custom targeted-gene expression profiling panel (Nanostring platform) to assess the mRNA abundance of all BCL-2 family members. In line with the observed effects of BCL-2 overexpression on rRNA synthesis rate, we confirmed an inverse correlation between BCL-2 mRNA levels and nucleolar area in both cohorts. This negative correlation was specific, since among all BCL-2 family members, only BCL-2 correlated with nucleolar area. Furthermore, nucleolar area was an independent prognostic predictor in multivariate analysis, outperforming BCL-2 mRNA levels.

These data indicate that the adverse prognostic influence related to BCL-2 overexpression could be due, in relevant part, to a reduced baseline rRNA synthesis rate, which in turn impairs p53-mediated response to chemotherapy, providing the rationale for novel, specific combination strategies aimed at overcoming BCL-2 induced chemoresistance in DLBCL.