Cell differentiation stage drives mechanisms of apoptosis evasion in Waldenström macroglobulinemia Free

Waldenström Macroglobulinemia (WM) is an incurable lymphoid malignancy characterized by a spectrum of differentiation states from clonal B lymphocytes (Ly) to IgM-expressing plasma cells (Pc). Activation of mitochondrial apoptosis by Bcl-2 antagonists is already under clinical investigation for treatment-naïve (TN) and relapsed/refractory (R/R) patients. However, the biological mechanisms whereby WM cells evade mitochondrial apoptosis remain undefined. In this work, we leveraged functional and single-cell transcriptomic approaches to unravel fundamental principles of apoptosis evasion in WM.

We collected viable WM cells from the bone marrow of 27 WM patients (25 MYD88^L265P and 2 MYD88^WT; 25 patients were TN, 2 were R/R). Samples were subjected to intracellular BH3 profiling (iBH3), a functional assay that derives the apoptotic priming and the anti-apoptotic dependencies based on the pattern of cytochrome c release in response to pro-apoptotic stimuli. Surface staining with anti-CD19 and anti-CD138 antibodies was included to discriminate Ly and Pc in each sample. We found that the apoptotic profile differed considerably between malignant Ly (CD19⁺CD138^-) and Pc (CD19⁺CD138⁺). WM Pc displayed lower apoptotic priming (P=0.002) and lower Bcl-2 dependency (P=0.03) than WM Ly. In contrast, non-Bcl-2 anti-apoptotic dependencies, particularly Mcl-1 dependency, were higher in Pc than Ly (Mcl-1 dependency: 93.0 and 42.8%, respectively, P<0.001).

Despite being predominantly Bcl-2 dependent, Ly showed remarkable heterogeneity across samples. Integrating Ly apoptotic profiles with patient clinical data, we found a positive correlation between the amount of serum monoclonal component (MC) and co-dependency upon Mcl-1 specifically detected in Ly (Pearson r=0.68, P<0.001). We also observed an increasing gradient of Mcl-1 dependency (P<0.001) when comparing Ly from patients affected by chronic lymphocytic leukemia (a prototypical Bcl-2 dependent malignancy, N=17), hyposecretive (MC<30 g/L, N=20), and hypersecretive WM (MC<30 g/L, N=7).

To assess whether Ly and Pc express different sets of pro- and anti-apoptotic genes, we performed single-cell RNA sequencing coupled with BCR sequencing on 8 WM bone marrow samples, 3 of which were hypersecretive. As expected, BCL2 was upregulated in WM Ly compared to residual normal memory B-cells. With respect to normal Pc, WM Pc expressed a higher level of the MCL1/PMAIP1 couple and a lower level of the pro-apoptotic BCL2L11 (encoding Bim). Focusing on tumor cells only, we identified 9 clusters with different transcriptional profiles. Five of them (Clusters 1-5) covered the Ly population, two harbored features of intermediate lymphoplasmacytic states, one corresponded to canonical JCHAIN^highPRDM1^high Pc, and an additional minor cluster was composed of atypical JCHAIN^highPRDM1^neg Pc. Consistent with iBH3 results, there was a clear-cut progressive increase of MCL1 expression during the differentiation of WM Ly towards canonical WM Pc. In contrast, BCL2, as well as BIRC3 and BIRC6, remained high up until the intermediate clusters and then dropped down at the Pc end of the differentiation trajectory. Interestingly, PMAIP1 followed a wave-like pattern with very high expression in intermediate states and low levels on the two ends of the differentiation process. BCL2A1, an anti-apoptotic gene often involved in therapy resistance, was highly expressed in a fraction of WM Ly belonging to Cluster 5. More importantly, within the WM Ly population, the highest expression of MCL1, at levels comparable to those observed in intermediate states,was detected in Cluster 4. This Ly cluster was particularly enriched in hypersecretive patients and may underpin the functional Mcl-1 dependency associated with this clinical phenotype.

Lastly, we searched for targetable signaling pathways transcriptionally upregulated in clusters with high MCL1expression. While B-cell receptor signaling and NF-kB activity were comparable across clusters, IRE1/XBP1 pathway and, to a lesser extent, STAT3 activity were increased in JCHAIN^highPRDM1^high Pc and may represent actionable targets to suppress Mcl-1 addicted cells.

In sum, cell differentiation stage coordinates the expression of Bcl-2 family genes and modulates anti-apoptotic dependencies in WM. Such transcriptional and functional intratumor heterogeneity may underlie the relatively low rate of deep responses to Bcl-2 antagonism in WM and suggest novel combination strategies.