Single-cell landscape of PBMCs in patients with IBR and IBS CLL
To study PBMC heterogeneity in CLL patients treated with ibrutinib and the underlying mechanism of ibrutinib resistance, we collected PBMCs from three IBR, four IBS CLL patients and two healthy donors and performed scRNA-seq using a droplet-based 10× Genomics platform. After quality control, we obtained 42,288 cells with a mean number of 4,760 cells per sample. The median number of unique molecular identifiers and genes per cell were 3,570 and 1,108 respectively. T-distributed stochastic neighbor embedding (t-SNE) visualization of filtered cells following integration and unsupervised clustering, revealed that cells from IBR and IBS samples were differently distributed in clusters, suggesting that IBR cells display a unique transcriptional pattern (Fig. 1A). To further differentiate these cells, we sorted them into 36 clusters (Fig. 1B) and annotated them as five major categories, specifically CD79A+ B-cells, CD3E+ T-cells, KLRD1+ NK-cells, CD14+ monocytes, and CD1C+ dendritic cells (Fig. 1C, D; Fig. S1).
First, we calculated the proportion of cell types in each sample (Table S2). B cells exhibited significantly increased expansion in CLL patients compared to that in healthy donors. (Fig. 1E). As copy number variation (CNV) is a common phenomenon in CLL , we predicted InferCNV in IBR and IBS patients using scRNA-seq data Fig. 1F). We found that one IBR patient (R3) carried a deletion of the chromosome 8 short arm, consistent with the findings of Burger . Two IBR patients (R2 and R3) carried a deletion of the chromosome 9 long arm and one IBR patient (R3) carried a deletion of the chromosome 4 short arm, implying that del(9q) and del(4p) might be associated with ibrutinib resistance. Obviously, we found that chromosome 6p was often amplified, consistent with the results of Brown .
B cells from IBR patients exhibit a unique transcriptional pattern
Next, we investigated B-cell heterogeneity in IBR and IBS patients by re-clustering the 30,417 identified B cells into 21 clusters (Fig. 2A). B cells from IBR patients exhibited different distributions compared with those from IBS patients. CytoTRACE analysis revealed that B cells in IBR CLL patients had higher stem index scores, implying that abnormal B cell stemness might be involved in the resistance to ibrutinib (p < 2.2e-16, Fig. 2B and 2C).
According to the proportion of B cells from IBR samples, B-cell clusters were categorized into three main subgroups, namely IBR (> 50 % of B cells from IBR samples) and IBS (> 70 % of B cells from IBS or NC samples). Other clusters were defined as shared clusters and clusters with < 200 cells were filtered (Fig. 2D). To elucidate the functional differences between IBR and IBS/shared clusters, we performed metabolic enrichment analysis. IBR clusters were significantly enriched in glycolysis and gluconeogenesis, which supply energy and support cancer cell growth (Fig. 2E, Table S3). We also found that many glycometabolism pathways, such as the pentose phosphate pathway, pentose and glucuronate interconversion, and fructose and mannose metabolism, were enriched in IBR clusters. Further, we found that glutathione metabolism was enriched in IBR clusters, consistent with Zhang who reported that elevated glutathione levels can increase leukemia cell survival and protect them against drug-induced cytotoxicity . Together, these findings suggest that B cells from IBR patients exhibit a unique transcriptional and metabolic pattern compared with B cells from IBS patients.
Difference between intercellular interactions in the microenvironment of IBR and IBS cells
Intercellular communication mediated by ligand–receptor complexes is essential for coordinating biological processes, such as differentiation and inflammation. Therefore, we studied the interaction between B cells and other types of cells from IBR and IBS clusters by performing cell–cell communication analysis using CellphoneDB. IBR-B cells displayed more interactions with monocytes, NK, T, and dendritic cells than IBS-B cells, suggesting that IBR-B cells could actively build connections with other cells to reshape the protective niche, which would be beneficial for cell survival (Fig. 2F). Previous studies have shown that co-culturing with stromal NK cells can increase the oxidative phosphorylation of CLL cells, promoting their proliferation . Kurtova, et al.  also found that bone marrow stromal cells provide survival and drug resistance signals for CLL cells against spontaneous and drug-induced apoptosis.
Notably, the LGALS9–CD47 interactions only existed between T cells and IBR-B cells, and not IBS cells (Fig. 2G, Table S4), which has not previously reported in CLL. We also found that other B cell–T cell interactions such as MIF-TNFRSF14 and LILRA4-BST2 were specifically enriched in IBR clusters. These findings indicated that IBR B cells might reprogram intercellular interaction patterns in CLL patients. Therefore, the interaction between CLL cells and other microenvironment cells might be involved in the development of drug resistance.
LGALS1 and LAG3 are associated with the transition between IBS and IBR
Although genome-level features have been extensively studied in IBR patients with CLL [35, 36], the transcriptional characteristics are rarely reported. To improve our understanding of the underlying mechanism of ibrutinib resistance, we performed bulk RNA sequencing with PBMCs from 6 IBR and 40 IBS CLL patients. Ninety genes were significantly upregulated in the IBR group compared to levels in the IBS group, among which LGALS1, LAG3, and PTMS were the top three upregulated genes (Fig. 3A, Table S5). In addition, we identified highly expressed genes in each IBR cluster at the single cell level (719 genes; Table S6), including LGALS1, LAG3, and PTMS. Moreover, we investigated the developmental trajectory of B cells in IBR and IBS patients using pseudo-time analysis. Remarkably, B cells from IBR samples were distributed at the end of one state track, indicating that IBR cells might evolve from IBS cells (Fig. 3B). According to the pseudo-time analysis, we some genes were found to be gradually upregulated along the trajectory from IBS to IBR (Fig. 3C). LGALS1 was highly expressed in five single-cell IBR clusters (Fig. 3D). LGALS1 and LAG3 expression increased along the transition trajectory, whereas PTMS did not coincide with this trend (Fig. 3E). Interestingly, PTMS and LAG3 were found to be located adjacent to each other on chromosome 12p13.31 and bulk-RNA data indicated the co-expression of these genes.
LGALS1 and LAG3 typically act as immune checkpoints that repress innate and adaptive immune programs [37–39]. To further elucidate the function of LGALS1 and LAG3 in CLL patients, we analyzed the bulk RNA-seq data. Genes co-expressed with LGALS1 were identified, including S100A4, S100A6, and COX5B, which are associated with cancer cell invasion and proliferation  (Fig. 3F, Table S7), as well as genes co-expressed with LAG3, such as HSPG2, which is associated with poor prognosis in acute myeloid leukemia  (Fig. S2, Table S8).
GO analysis revealed that genes co-expressed with LGALS1 were significantly enriched in mitochondrial ATP synthesis-coupled electron transfer and oxidative phosphorylation (Fig. 3G, Table S9). LGALS1 might play an important role in the proliferation of CLL cells. In contrast, genes co-expressed with LAG3 were enriched for pathways related in glucose metabolism (Fig. S3). Together, LGALS1 and LAG3 are related to the metabolic pathways and might be involved in cell resistance to ibrutinib.
High expression of LGALS1 and LAG3 closely correlates with poor outcome in CLL
Next, we detected and analyzed the overall survival (OS) of CLL patients, finding that patients with higher LGALS1 and LAG3 expression showed poorer OS (Fig. 4A, left and middle). Moreover, Kaplan-Meier analysis demonstrated that patients with concurrent high expression of LGALS1 and LAG3 exhibited a worse OS compared to those with high LGALS1 or LAG3 expression respectively (Fig. 4A, right). To further validate the survival analysis results, we also analyzed GSE22762 data from the GEO database  which is comprise of 107 CLL patient samples (Fig. 4B). As expected, these results both demonstrated that an LGALS1 and LAG3 gene panel is associated with poor prognosis for CLL patients.
To evaluate and confirm the clinical relevance of LAG3 and LGALS1 in CLL, we analyzed their expression and the clinical features of CLL patients (Fig. 4C). Patients with higher level of LAG3 and LGALS1 were found to have higher mutation rates for SF3B1 (p = 0.035) or NOTCH1 (p = 0.038). Immunoblots were then performed to compare protein levels between IBS and IBR patients, and the results revealed higher levels of Gal-1 and LAG3 in IBR patients (Fig. 4D, Fig. S4A). As Gal-1 is a secretory protein, we determined its plasma concentration as well as that of LAG3 in 16 treatment-naïve, refractory and/or relapsed (R/R) CLL patients using ELISA. As expected, Gal-1 and LAG3 levels were significantly higher in R/R CLL patients (Fig. S4B). Collectively, these results indicate that elevated expression of LGALS1 and LAG3 is associated with poor prognosis in CLL.
LGALS1 and LAG3 are upregulated in the established ibrutinib-resistant cell line
As LGALS1 and LAG3 indicated poor prognosis in CLL patients, we sought to explore the reasons for this with the acquired ibrutinib-resistant cell line (MEC1-IR, Fig. 5A). CCK8 assays indicated that MEC1-IR cells were significantly more resistant to ibrutinib than the parental cells (Fig. 5B). Annexin-V/PI staining also revealed a pronounced increase in cell death in MEC1 cells (Fig. 5C, D). Next, we examined the expression of LGALS1 and LAG3 in parental and resistant cells, revealing that they are markedly higher in MEC1-IR cells (Fig. 5E). Expectedly, immunoblotting and ELISA showed consistent results (Fig. 5F-H). These results demonstrate that LGALS1 and LAG3 are upregulated in IBR cells.
The Gal-1 inhibitor OTX008 effectively inhibits the proliferation of CLL cells
Recent studies have demonstrated the efficacy of OTX008, a Gal-1 inhibitor, in preclinical models of multiple tumors . Therefore, we studied the anti-tumor effect of OTX008 on IBS and IBR CLL cells. First, we examined the expression of Gal-1 and LAG3 with OTX008 treatment. We found that OTX008 could decrease LAG3 mRNA levels (Fig. 6A). Meanwhile, the protein levels of Gal-1 and LAG3 were decreased in a dose-dependent manner in both CLL cell lines and primary cells (CLL-1) (Fig. 6B). This observation was validated by immunofluorescence (Fig. 6C). Though MEC1-IR cells displayed higher proliferation than MEC1 cells, the proliferative capacity of both groups was almost completely inhibited by OTX008 (Fig. 6D). Subsequently, flow cytometric analysis revealed that MEC1-IR cells underwent markedly increased apoptosis compared to that in MEC1 cells (Fig. 6E). Primary CLL cells were then treated with OTX008 and trypan blue staining revealed that OTX008 was more effective against the IBR group than IBS (Fig. 6F). Overall, our findings demonstrate the potential clinical utility of OTX008 for CLL, and particularly for IBR patients.