Chronic Lymphocytic Leukemia (CLL) is a mature B cell malignancy noted for its clinical and biological heterogeneity (Delgado et al., 2020). This variability stems from a complex interaction between genetic factors, epigenetic modification, and the tumor microenvironment (TME). A subset of patients undergoing treatment may experience transformation into an aggressive form of lymphoma, a progression known as Richter Transformation (RT) (Wang and Ding, 2020). Key drivers of RT involve alterations in critical cellular pathways including the cell cycle, MYC, NOTCH and NF-κB pathways (Nadeu et al., 2022). Transcriptomic analyses have revealed that RT cells exhibit activation of the oxidative phosphorylation pathway alongside a downregulation of B-cell receptor (BCR) signaling (Nadeu et al., 2022). In CLL, activation of the BCR is known to upregulate MYC expression via a BTK-dependent mechanism (Yeomans et al., 2016). Increased MYC target gene expression was found in lymph nodes with corresponding increases in MYC protein levels in both lymph nodes and unmutated CLL (U-CLL) (Herishanu et al., 2011). While genetic events in MYC are rare in CLL (Huh et al., 2008), the transcriptomic activation of MYC remains unexplored. Therefore, this study aims to explore the activation of MYC target genes across various stages of CLL—including diagnosis, progression, relapse, and RT and various disease subgroups (e.g., unmutated IGHV-CLL cases (U-CLLs), genetic alterations).

We used a comprehensive dataset comprising 263 bulk RNAseq CLL samples from the ICGC cohort without genetic alterations in MYC (Knisbacher et al., 2022), along with 12 samples from 6 CLL patients pre- and post-RT (Nadeu et al., 2022). Additionally, we analyzed single-cell RNAseq data from 40,725 cells including CLL, TME and RT (Nadeu et al., 2022). A 285-gene MYC target gene signature was created by integrating data from various sources (Methods, Supplementary Figure S1; Supplementary Table S1).

We initially characterized the activation of MYC target genes across clinicobiological subgroups of CLL (Supplementary Figures S2a–c). Our findings show a significant increase in MYC target gene activation in U-CLLs compared to mutated IGHV CLLs (M-CLLs) (p < 0.001) (Figure 1a). In terms of gene mutations (Supplementary Figure S2b), cases with HMCN1 mutations showed lower MYC target gene activation (Supplementary Figure S2b). Additionally, CLL cases with trisomy 12 exhibited higher levels of MYC target gene activation than those without trisomy 12, regardless of IGHV mutational status (p < 0.001) (Figure 1b; Supplementary Figure S2c). Considering the clinical trajectory of CLL, particularly in RT, we observed a statistically significant upregulation of MYC target genes in RT compared to pre-RT stages (p = 0.015) (Figure 1c). MYC target gene activation showed a strong positive correlation with expression levels of MYC in CLL phase (Supplementary Figures S2d,e).

Given the multifaceted role of MYC activation, we analyzed its correlation with other pathways to deepen our understanding of its impact in CLL and RT phase (FDR < 0.01 and |r| = 0.3) (Figure 1d). In the CLL phase, MYC activation correlated with most of the pathways (45 out of 51 examined), aligning with its known pleiotropic function (Das et al., 2023). Positive correlation was observed between BCR signaling and MYC activation in CLL, as expected, validating our methodology (Yeomans et al., 2016). Conversely, in the RT phase, only 19 pathways showed a correlation with MYC target gene activation. Notably, a stronger correlation was observed between cell cycling and MYC target gene activation in the RT phase, diverging from its broader functional role as seen in CLL. Interestingly, MYC target gene activation in RT appeared to be independent of the BCR signaling pathway (negative correlation observed), consistent with literature suggesting a downregulation of this pathway in RT (Nadeu et al., 2022). This lack of association between MYC target genes and BCR signaling in RT suggests that MYC may support CLL cell survival via alternative cellular programs, distinct from traditional BCR-mediated mechanisms. Additionally, we noted a loss of correlation between the TGF-beta signaling pathway and MYC target gene activation in RT, aligning with previous studies that highlight the diminution of this pathway in RT (Auge et al., 2020).

Next and based on the score of MYC target gene activation, we stratified the CLL cases into high, intermediate, and low activation groups based on quartiles of the score values (Figure 1e). Notably, the high activation group includes both M-CLL (n = 40) and U-CLL (n = 49) pre-treatment CLL cases (Supplementary Table S2). Kaplan-Meier curve analysis revealed significant differences among the three MYC groups, with high MYC target gene activation associated with a significantly shorter time to first treatment (p < 0.001), an effect that remained independent of IGHV mutational status. (Figure 1f; Supplementary Figure S2f). Differential expression analysis between the high and low MYC activation groups identified 370 genes (FDR < 0.01) (Figure 1g). Pathway analysis (p < 0.05) linked MYC activation to key components of the TME interactions such as ‘TNFα signaling via NFκB’ (enriched genes: EGR1, IL1B, KLF10, SOCS3, CCL4, CDKN1A, FOS, FOSB, NR4A1, BCL2A1, DUSP4, NR4A3, ICAM1, SGK1, ID2, MYC, GADD45A, MARCKS, FOSL2, PTGS2) and ‘IL6 JAK STAT3 signaling’ (enriched genes:ITGA4, IL1B, SOCS3, CD38) (Figure 1h). To gain insights into the role of MYC target gene activation in TME interactions we examined 27,837 malignant cells and 3,715 cells from the TME (comprising T, and myeloid cells) in CLL phase and 7,479 cells after RT and 1694 TME derived from four samples (Figures 2a,b; Supplementary Figures S3a,b). UMAPs of CLL and RT cells were able to distinguish the TME from malignant cells, however the CLL cell clustering showed high donor specificity (Figures 2a,b).

MYC target gene activation represents the principal trajectory of CLL cell orientation across donors, as visualized in the UMAP plots (Figures 2a,b). Notably, in the CLL phase, this trajectory is independent of cell cycle activity (Pearson r = 0.04, Supplementary Figure S3c) highlighting the importance of MYC target gene activation in the transcriptomic profiles of the cells. In the RT phase MYC activation was higher compared to CLL and the orientation of the cells was influenced by cell cycle as well, further supporting the bulk RNAseq analysis (Pearson R of MYC target gene activation and S.Score = 0.39) (Supplementary Figures S3d,e).

High MYC target gene activation was associated with increased TME interactions in both CLL and RT phase especially with myeloid cells (Figures 2c,d). There is a notable decrease in interactions from the high to intermediate and low activation groups highlighting the role of MYC in the TME communication. Recent studies suggest the MYC as a master regulator in host immune response against the cancer but mainly focusing on the mutational activation of MYC (Dhanasekaran et al., 2022). The top10 interactions in each comparison showed unique interactions with the TME for high MYC target gene activation groups in both CLL and RT phase The RT phase exhibited greater interaction specificity (max = 0.4 in CLL, max = 0.6 in RT) (Figures 2e,f). Interactions with myeloid cells revealed BAFF-R in both phases but different members (TNFRS13C in CLL and TNFRSF17 in RT), with higher interaction specificity in high MYC target gene activation. In CLL, ICAM1/IL2RA and CXCL8/CD79A interactions showed high specificity for high MYC target gene activation (Figure 2e). CD79A interactions were not detected in RT, which may be related to the observed downregulation of the BCR signaling pathway in silico. RT showed a different set of interactions with microenvironment, notably through TLR9 (Figure 2f). TLR9 emerged as a remarkable example of alternative survival pathways post-relapse and RT, promoting CLL cell migration and survival. Considering a recent publication reported high efficacy for combined targeting of TLR9 and BTK (Kennedy et al., 2021), our study suggests that this combination could indeed enhance treatment efficacy and potentially prevent RT. Lower interaction specificity was observed with T cells (Supplementary Figures S3f,g).

Numerous interactions among malignant cells, especially with high MYC target gene activation cells, were found, including BAFF-R/CD70, CD70/CD27, and BST2/LILRA4 (Figure 2g; Supplementary Figure S3h). Most of these interactions occurred in the RT phase with higher specificity, suggesting an antigen-independent cell-autonomous signaling that leads to MYC activation and proliferation, creating a feedback loop that reinforces the malignant phenotype. Our findings, which indicate that higher MYC target gene activation is associated with increased interactions with TME, and that high MYC levels facilitate communication with other chronic lymphocytic leukemia (CLL) cells, were validated using data from 13,280 cells including both CLL and TME before ibrutinib treatment from the Rendeiro et al. (2020) (Supplementary Figures S4a,b). Furthermore, MYC target gene activation was observed to gradually decrease during ibrutinib treatment, with analysis conducted on 10,299 CLL cells before ibrutinib and 9,450 CLL cells during ibrutinib treatment across three different time points (Supplementary Figure S4c). This finding underscores the dependency of MYC on BCR signaling in CLL.

Our study highlights the implication of MYC in CLL even in the absence of a genetic component. High MYC target gene activation was associated with U-CLLs and trisomy 12, treatment outcomes and RT. Additionally, high MYC transcriptomic activation showed increased interactions with the TME, suggesting MYC as an orchestrator of TME interactions thereby explaining its link to various cellular processes.

In RT, we observed high activation of MYC target genes that is independent of the BCR pathway. This activation is highly correlated with cell cycling and may derive survival signals from TLR9 interactions and malignant-malignant cell interactions. These findings support enhanced patient stratification by considering the transcriptomic activation of MYC, especially with new options emerging for targeting the previously “undruggable” MYC.

Thus, targeting MYC at the translational level (Largeot et al., 2023) represents an exciting opportunity to complement transcriptomic stratification and may provide therapeutic benefit in MYC-high CLL patients. This could be particularly valuable in the RT setting, where MYC activity is high but uncoupled from canonical BCR signaling. Future studies should investigate whether translational inhibitors could synergize with BTK or TLR9 inhibitors (Largeot et al., 2023), especially in high MYC activation states identified in the present study.