Decoding the Immune Microenvironment of Secondary Chronic Myelomonocytic Leukemia due to DLBCL with CD19 CAR-T Failure by Single-cell RNA-seq

doi:10.21203/rs.3.rs-5200819/v1

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Decoding the Immune Microenvironment of Secondary Chronic Myelomonocytic Leukemia due to DLBCL with CD19 CAR-T Failure by Single-cell RNA-seq

https://doi.org/10.21203/rs.3.rs-5200819/v1

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Multiple studies have confirmed the occurrence of second tumors as a rare incidence of CAR-T therapy, but one of the complications that does warrant in-depth exploration. According, given the relatively small number of reported second tumor types thus far, additional comprehensive occurrence and characterization of a new second tumor type after CAR-T therapy remains essential for understanding the risk of potential tumors with this therapy, as well as for defining the role of immune microenvironment in malignant transformation. In this article, a new second tumor type CMML was identified in a patient who had received CD19 CAR-T therapy for DLBCL. The immune microenvironment of both the pre- and post-treatment of secondary CMML and primary CMML were deeply profiled by ScRNA-seq. Our results demonstrated an enhanced inflammatory cytokines, chemokines, and immunosuppression state of monocytes/macrophages, which may inhibit the cytotoxicity of T/NKs in secondary CMML. In contrast, the cytotoxicity of T/NKs were enhanced in secondary CMML after treatment. Collectively, our results highlight a new type of second tumor, CMML after CAR-T therapy and provide a framework for defining the immune microenvironment of second tumor occurrence after CAR-T therapy. Our results also provide a rationale for targeting macrophages to strengthen CMML treatment.

Second tumor after CAR-T therapy

chronic myelomonocytic leukemia

immune microenvironment

DLBCL

ScRNA-seq

Chronic myelomonocytic leukemia (CMML), an aggressive clonal hematopoietic malignancy characterized by persistent monocytosis, combines myeloid cell proliferation with myeloid cell dysplasia and ineffective hematopoiesis. ^1–4 Prior work has demonstrated that CMML is extremely heterogenous in presentation and outcomes, which makes diagnostic, prognostic, and therapeutic decision-making challenging. ^5,6 Despite CAR-T cell therapy is effective against hematological cancers, a small percentage (0.9%-1.2%) of patients may develop second malignancies, such as MDS, AML, T-cell lymphomas and solid tumors after treatment with CD19-CAR-T. ^7–11 However, secondary CMML due to DLBCL with CD19 CAR-T failure (secondary CMML) has not yet been reported. Therefore, there is a need to study the underlying mechanisms of the occurrence and development of secondary CMML and to explore new potential therapeutic strategies.

Previous studies have used ScRNA-seq to analyze the diversity of HSCs/HSPCs. Meghan C. Ferrall-Fairbanks et al. identified three differentiation paths for HSPCs in CMML: monocytic, megakaryocyte-erythroid progenitor (MEP), and normal-like. ¹² Daniel H. Wiseman et al. analyzed 6,826 stem cells from CMML patients and healthy controls, finding that CMML stem cells showed higher expression of myeloid-lineage and cell cycle genes, and lower expression of genes specific to normal HSCs.¹³ Guillermo Montalban-Bravo showed that CMML progression starts from immature stem cell-level myeloid progenitors, with downstream monocytes adapting to resist therapy and hasten disease. ¹⁴. Despite these findings, our understanding of the BM immune microenvironment of CMML is still not complete, especially in secondary CMML.

In the present study, we reported a new second tumor type, CMML after CD19 CAR-T therapy and performed ScRNA-seq for CD45⁺ BM cells for the patient both at pre- and post-treatment status as well as primary CMML. We comprehensively characterize the cellular and molecular alterations in the BM immune microenvironment in HCs, primary CMML, and secondary CMML and show the underlying mechanisms in secondary CMML, as well as resolve the alterations in the BM microenvironment of secondary CMML after treatment with Azacitidine plus Venecla.

The detailed materials and methods can be found in supplementary materials.

3.1. Morphological and immunophenotypic differences between primary and secondary CMML

To identify the potential heterogeneities of secondary CMML and primary CMML, we analyzed the morphology and immunophenotypes of secondary CMML and primary CMML. Our results indicated that secondary CMML present distinct morphology and immunophenotypes compared with primary CMML (Supplementary Fig. 1A, B and C, Supplementary Table 1). In conclusion, there may be important differences between primary CMML and secondary CMML, as shown by the results of the morphological and flowcytometry immunophenotyping analyses.

3.2. Bone marrow immune microenvironment features of primary CMML, and secondary CMML before treatment

To generate a single-cell transcriptome landscape of BM CD45⁺ cells in primary CMML, secondary CMML, and HCs, we collected fresh BM samples from patients with primary CMML and secondary CMML. Subsequently, ScRNA-seq was performed on the CD45⁺ BM cells (Fig. 1A). The ScRNA-seq of BM samples of HCs were obtained from You et al. ¹⁵ After stringent quality control, a total of 37,011 cells were obtained from four BM CD45⁺ samples and further classified into seven cell lineages (Fig. 1B and C, Supplementary Table 2). Based on the expression of canonical marker genes (Fig. 1D and Supplementary Fig. 2), we defined them as monocytes/macrophages (CD68, CD14), HSCs/HSPCs (CD34, ERG), granulocytes (MPO), T/NK cells (CD3D, CD3E, NKG7, KLRB1), DCs (TMP2, LILRA4), GMPs (CD38, KIT, PPBP), and B cells (CD79A, CD19). Significantly, patients with secondary CMML had the lowest ratio of T/NK cells and the highest ratio of monocytes/macrophages (Fig. 1E). This finding was also supported by an increased expression of monocytes/macrophages-related genes such as LYZ, CD14, MAFB, and MNDA and a decreased expression of T/NK cell differentiation-related genes CD52, KLRB1, TRAC, NKG7, GZMA, GZMB, and GNLY (Fig. 1F). Patients with secondary CMML exhibited heightened infection, inflammation, and phagocytosis pathways (Fig. 1G), alongside reduced T and B cell receptor signaling (Fig. 1H). This suggests that secondary CMML development may be linked to impaired T/B cell immune responses, leading to secondary infections and subsequent activation of monocytes/macrophages, causing systemic inflammation and contributing to CMML progression.

3.3. Increased infection-mediated phagocytosis, chemotaxis and immune responses of monocytes/macrophages in secondary CMML

We next divided monocytes/macrophages into 11 subpopulations based on marker genes (Figs. 2A, B, and C, Supplementary Table 3). Cluster 0 showed a higher level of expression of GAS7, CSF2RA, and PPARG; Cluster 1 was characterized by higher expression levels of CD74, IFI30, and HLA-DRA; Cluster 2 was reported to express high levels of CD14, MNDA, and SERPINB1; Cluster 3 expressed higher levels of FCGR3B, CSF3R, and MALAT1; Cluster 4 expressed high levels of FCGR3A, SIGLEC10, CX3CR1, and LILRB2; Cluster 5 expressed higher levels of CXCL8 and S100A8; Cluster 6 demonstrated higher levels of expression of ITGAM, CD300E, and CD4; Cluster 7 showed a higher level of expression of IFIT3, IFI44L, IFIT1, ISG15, and IFI44; Cluster 8 showed high levels of expression of HAVCR2 and MYO9B; Cluster 9 expressed high levels of ICAM1, IL1B, and TNF; and Cluster 10 was reported to express high levels of HLA-DOA, ID3, HLA-DQA2, CD2, CD1C, and ID1 (Fig. 2D, Supplementary Fig. 3). Interestingly, the secondary CMML patient demonstrated an increased percentage of Clusters 1, 2, 3, 7, and 10 but a decreased percentage of Clusters 0, 4, 5,6, 8, and 9 (Fig. 2E). The upregulated KEGG pathways enriched for DEGs in Clusters 1, 2, 3, 7, and 10 included antigen processing and presentation, coronavirus disease (COVID-19), oxidative phosphorylation, chemokine signaling pathway, a neurotrophin signaling pathway, endocytosis, Fc gamma R-mediated phagocytosis, and influenza A. However, Cluster 0, which has upregulated KEGG pathways, including ubiquitin mediated proteolysis, thyroid hormone signaling pathway, and MAPK signaling pathway, was only found in primary CMML. The upregulated KEGG pathway in Clusters 4, 5, 6, 8, and 9 included Th1 and Th2 cell differentiation, a T cell receptor signaling pathway, PD-L1 expression and a PD-1 checkpoint pathway in cancer, an Epstein–Barr virus infection, lipids and atherosclerosis, an NF-kappa B signaling pathway, and a Kaposi sarcoma-associated herpesvirus infection (Fig. 2F). Collectively, these results indicated that monocytes/macrophages in secondary CMML before treatment correlated with an infection-mediated phagocytosis, chemotaxis and immune responses microenvironment, which may contribute to the development of CMML.

3.4. Transcriptomic differences of the monocytes/macrophages between primary CMML and secondary CMML

We next elucidated the transcriptomic differences between the monocytes/macrophages of primary CMML and secondary CMML. In total, the overall DEG analysis showed that 1673 genes were upregulated, whereas 582 were downregulated in secondary CMML compared to HCs (Supplementary Fig. 4A, Supplementary Tables 4 and 5). The KEGG pathway enrichment of upregulated genes in secondary CMML included salmonella infection, pathogenic Escherichia coli infection, human cytomegalovirus infection, yersinia infection, tuberculosis, viral carcinogenesis, phagosomes, lysosomes, and Fc gamma R-mediated phagocytosis (Supplementary Fig. 4B). In contrast, the KEGG pathway enrichment of downregulated genes included coronavirus disease (COVID-19), oxidative phosphorylation, antigen processing and presentation, and viral myocarditis (Supplementary Fig. 4C). Specifically, the expression levels of genes encoding proteins known to be important for the KEGG pathways were significantly higher in secondary CMML (Supplementary Fig. 4D). Subsequently, an analysis of the DEGs of patients with secondary and primary CMML showed that 1588 genes were upregulated, whereas 1834 were downregulated in secondary CMML compared to primary CMML (Supplementary Fig. 4E, Supplementary Tables 6 and 7). The KEGG pathway enrichment of upregulated genes in secondary CMML patient included salmonella infection, phagosomes, coronavirus disease (COVID − 19), pathogenic Escherichia coli infection, oxidative phosphorylation, and yersinia infection (Supplementary Fig. 4F). In contrast, the KEGG pathway enrichment of downregulated genes included chronic myeloid leukemia, T cell receptor signaling, a chemokine signaling pathway, an Epstein–Barr virus infection, and a Kaposi sarcoma-associated herpesvirus infection (Supplementary Fig. 4G). Specifically, the expression levels of genes encoding proteins known to be important for the KEGG pathways were significantly higher in secondary CMML (Supplementary Fig. 4H). These findings collectively suggest that increased infection in secondary CMML may correlate with the occurrence of this disease.

3.5. Decreased cytotoxicity of T/NK cells in patients with secondary CMML before treatment

We further identified subclusters in the T/NK cells. A total of 16 subpopulations were identified based on marker genes (Figs. 3A, B, C, and D, Supplementary Table 8). Based on gene expression profiling, isolated cells from the BM were then categorized into 11 main subclusters, including three subtypes of CD4⁺Naïve T (Tn, Clusters 0, 5, and 8), four subtypes of early effector memory T cell (early Tem, Clusters 1,2,3 and 9), one subtype of Temra (Cluster 6), one type of NK-like cells (Cluster 7), one subtype of Tc17 (Cluster 10), one subtype of γδ-T cells (Cluster 11), one subtype of Treg (Cluster 13), one subtype of Tcm (Cluster 14), and one subtype of proliferating T cells (Cluster 15) (Fig. 3E, Supplementary Table 8). Cluster 13 included T cell markers and myeloid cell markers. Interestingly, the percentages of Clusters 0, 1, and 15 were increased in secondary CMML (Fig. 3F). After merging the same cell types, we found that the percentages of early Tem, proliferating T cells, Tcm, and Treg increased, while the percentage of γδ-T cells, NK-like cells, NKT cells, and Tn cells decreased, which may also contribute to the occurrence and development of secondary CMML (Fig. 3G).

3.6. Transcriptomic differences of T/NK cells between the primary CMML and secondary CMML

We also analyzed transcriptional differences in T/NK cells of patients with primary CMML and secondary CMML. A DEG analysis indicated that 589 genes were upregulated, whereas 694 were downregulated in secondary CMML, compared to HCs (Supplementary Fig. 5A, Supplementary Tables 9 and 10). The KEGG pathway enrichment of upregulated genes in patients with secondary CMML included a salmonella infection, a human papillomavirus infection, an Epstein–Barr virus infection, viral carcinogenesis, a human T-cell leukemia virus 1 infection, and a Yersinia infection (Supplementary Fig. 5B). In contrast, the KEGG pathway enrichment of downregulated genes included natural killer cell-mediated cytotoxicity, and a T cell receptor signaling pathway (Supplementary Fig. 5C). Specifically, the expression levels of genes encoding proteins known to be important for the KEGG pathways were significantly higher in secondary CMML (Supplementary Fig. 5D). Subsequently, an analysis of the DEGs of patients with secondary CMML and with primary CMML showed that 1314 genes were upregulated, whereas 969 were downregulated in secondary CMML compared to primary CMML (Supplementary Fig. 5E, Supplementary Tables 11 and 12). The KEGG pathway enrichment of upregulated genes in secondary CMML included salmonella infection, phagosomes, oxidative phosphorylation, coronavirus disease (COVID-19), and leukocyte transendothelial migration (Supplementary Fig. 5F). In contrast, the KEGG pathway enrichment of downregulated genes included T cell receptor signaling, osteoclast differentiation, Th1 and Th2 cell differentiation, and Th17 cell differentiation (Supplementary Fig. 5G). Specifically, the expression levels of genes encoding proteins known to be important for the KEGG pathways were significantly higher in secondary CMML (Supplementary Fig. 5H). Taken together, secondary CMML patients have reduced tumor killing activity of T/NK cells in the BM microenvironment.

3.7. The potential indirect mechanisms of secondary CMML via cell–cell communication analysis

To study cell–cell communication in secondary CMML, we compared its signaling pathways with those of primary CMML. Figures 4A and B show that secondary CMML has fewer but stronger interactions than primary CMML. We then analyzed the signaling pathways in each cell population of both primary and secondary CMML. Figure 4C indicates that the overall signaling pathways in secondary CMML included IL16, CXCL, CCL, TNF, PARS, IL1, TRAIL, CD40, BTLA, CSF, GAS, IL2, TIGIT, and others. The incoming signaling patterns of secondary CMML included TIGIT, IL16, CXCL, IL1, TRAIL, CSF, GAS, IL2, and others so on (Fig. 4D). The outgoing signaling patterns of secondary CMML included TIGIT, IL16, CXCL, IL1, TRAIL, PVR, GAS, IL2, and others (Fig. 4E). We then performed a comparative analysis of secondary CMML and HC cell communication differences, and the results are consistent with the above (Supplementary Figs. 6A–E). Collectively, our results suggest that the monocytes/macrophages from secondary CMML are associated with inflammatory cytokines, chemokines, and immunosuppression, which may induce T/NK dysfunction.

3.8. The remodeled BM immune cells in secondary CMML post Azacitidine plus Venecla treatment

Azacitidine in combination with Venetoclax is often used in the treatment of CMML. ^16–18 To understand the effects of Azacitidine plus Venecla on secondary CMML patients, we analyzed immune cell cluster changes pre- and post-treatment. CD45⁺ cells were categorized into T/NK cells, monocytes/macrophages, B cells, CMPs, granulocytes, DCs, and HSCs (Fig. 5A). Figures 5B and 5C display cells from both the before-treatment and after-treatment groups. Post-treatment, the patient showed a higher percentage of T/NK cells and a lower percentage of monocytes/macrophages and granulocytes (Fig. 5D). In line with this result, most of the top 10 DEGs before treatment are related to monocytes/macrophages (e.g., VCAN, LYZ, DUSP6), while most of the top 10 DEGs after treatment are related to T/NK cells (e.g., IL32, IL7R, CCL5, Fig. 5E). The KEGG pathway enrichment of upregulated genes in the after-treatment group included antigen processing and presentation, natural killer cell-mediated cytotoxicity, and a T cell receptor signaling pathway (Fig. 5F). In contrast, the KEGG pathway enrichment of downregulated genes in the after-treatment group included lysosomes, phagosomes, and Fc gamma R-mediated phagocytosis (Fig. 5G). Overall, our results suggest that Azacitidine plus Venecla treatment remodeled the immune microenvironment of patients with secondary CMML towards an anti-tumor microenvironment.

3.9. The effect on monocytes/macrophages of Azacitidine plus Venecla treatment in secondary CMML

We next reclustered monocytes/macrophages and divided them into seven subpopulations (Fig. 6A). The monocytes/macrophages derived from the before-treatment or after-treatment group are shown in Figs. 6B and C. Interestingly, after treatment, the patient demonstrated an increased percentage of Clusters 0 and 6 but a decreased percentage of Clusters 1–5 (Fig. 6D). We then analyzed the top six DEGs of these seven subclusters. The marker genes of Cluster 0 are FCGR3B, MMP9, IFITM2, MME, CMTM2, and LITAF, and the marker genes of Cluster 6 are HLA-DQA1, HLA-DQB1, HLA-DPB1, HLA-DPA1, HLA-DRA, and CD74 (Fig. 6E). The KEGG pathway enrichment of upregulated genes in the after-treatment group included antigen processing and presentation, an IL17 signaling pathway, and cytokine–cytokine receptor interaction, which may contribute to the regulation of the T/NK cell cytotoxicity effect (Fig. 6F). In contrast, the KEGG pathway enrichment of downregulated genes in the after-treatment group included lysosomes, coronavirus disease (COVID-19), and efferocytosis (Fig. 6G). Collectively, these findings suggest that Azacitidine plus Venecla treatment for secondary CMML may improve the patient's BM immune microenvironment from a pro-tumor progression microenvironment to an anti-tumor immune microenvironment.

3.10. Sub-clustering of T/NK lineage in secondary CMML before and after Azacitidine plus Venecla treatment

We further identified the changes in T/NK cells post Azacitidine plus Venecla treatment. We divided the T/NK cells from the before-treatment group or the after-treatment group into 13 subpopulations (Fig. 7A). Cluster 0 was identified as NK-1 cells based on the expression of NKG7, GZMA, GZMB, and GZMH; Cluster 1 was identified as naïve T cells based on the expression of LTB, IL7R, EEF1A1, and TPT1; Cluster 2 was identified as proliferating T cells based on the expression of MKI67, PCLAF, STMN1, HMGB2, and TUBA1B; and Clusters 3 and 7 were identified as double cells based on non-T/NK markers expression (CSF3R, MNCA, NAMPT, S100A8, S100A9, S100A12, CST3, IFI30, LYZ, AIF1, LST1, and CTSS). Cluster 4 was identified as γδ-T cells based on the expression of TRDC, TRGC1, KLRC2, CCL4, and GNLY. Cluster 6 was identified as CD8⁺ cytotoxicity T cells based on the expression of CD8B, CCR7, RPS6, RPS5, and LEF1. Cluster 8 was identified as NEAT1-T cells based on the expression of NEAT1, RNF213, and MALAT1. Cluster 9 was identified as CCR9⁺T cells based on the expression of CCR9, CD59 and TRBC2 (Fig. 7B and Supplementary Table 13). The T/NK cells derived from the before-treatment group or after-treatment group are shown in Fig. 7C. Interestingly, the percentage of Naïve T 3, NK1, NK2, and proliferation T increased in the after-treatment group (Fig. 7D). The KEGG pathway enrichment of upregulated genes in the after-treatment group included a NOD-like receptor signaling pathway, natural killer cell-mediated cytotoxicity, a chemokine signaling pathway, cytokine-cytokine receptor interaction, a Toll-like receptor signaling pathway, and a T cell receptor signaling pathway, which demonstrates the anti-tumor features induced by Azacitidine plus Venecla treatment (Fig. 7E). After treatment, downregulated genes were enriched in pathways related to various infections and viral carcinogenesis, likely due to decreased monocytes/macrophages and increased T/NK cells (Fig. 7F). Overall, myeloid cells showed significant transcriptional changes post-treatment with Azacitidine plus Venecla, potentially affecting T/NK cell function in multiple ways.

3.11. Cell–cell communication analysis demonstrated that monocytes/macrophages are key players in secondary CMML post treatment with Azacitidine plus Venecla

Finally, we performed a cell–cell communication analysis on cells from a patient with secondary CMML post treatment with Azacitidine plus Venecla using CellChat software. Supplementary Figs. 7A and B demonstrate that the number of inferred interactions increased, while the interaction strength decreased in the secondary CMML group post treatment with Azacitidine plus Venecla compared to the number before treatment. Thereafter, we analyzed the overall signaling pathways of each cell population, both after and before treatment. Supplementary Fig. 7C indicates that the overall signaling pathways that correlate to monocytes/macrophages in the after-treatment group included apoptosis-related genes, THY1, ¹⁹ TRAIL, ²⁰ and APRIL, ²¹ which correlated with decreased numbers of monocytes/macrophages. Remarkably, other upregulated overall signaling pathways in the secondary CMML post Azacitidine plus Venecla treatment group are CD40 and CD23, which are considered to be T/NK cell activation molecules. ^22–24 The incoming and outgoing signaling patterns analysis in the after-treatment group indicated that similar results compared to overall signaling pathways analysis (Supplementary Figs. 7D and E).

In the present study, we reported secondary CMML in a patient with DLBCL for the first time. Our data show a significant increase in myeloid cells but a decreased percentage of T/NK cells in both primary and secondary CMML. The differentiation pathway of HSCs in CMML is more biased toward myeloid cells and less toward lymphoid differentiation, ^25,26 which explains the presence of more myeloid cells (monocyte/macrophages) and fewer T/NK cells in CMML patients. This finding is consistent with reported CMML disease features. ^27,28

Secondary CMML may arise from factors like TET2 mutation, MAS-L symptoms post-CAR-T cell therapy, and pathogenic infections. (1) The TET2 gene, crucial for DNA demethylation and gene regulation, is vital in hematopoiesis, affecting HSC self-renewal and monocyte differentiation. ²⁹ The sequence of molecular abnormalities is crucial, with primary clonal lesions showing main clinical features and later mutations indicating disease progression. TET2 and ASXL1 are often the primary clonal events in CMML patients.^30–32 In the present study, patients with secondary CMML carried a TET2-pure mutation (TET2: NM_001127208: exon3:c.C86G; p.P29R), which explains the occurrence of secondary CMML and increased monocytosis, at least in part. (2) Somatic mutations in CMML are heterogeneous and only partially explain the variability in clinical outcomes. ³³ Multiple studies have shown MAS-L or monocytosis/macrophagocytosis after CAR-T cell therapy. ^34,35 Myeloid-derived macrophages have been found to play a critical role in CRS pathogenesis, and these cells mediate the major production of core cytokines, including IL-6, IL-1β, and IFN-γ. These pro-inflammatory cytokines promote monocytosis/macrophagocytosis. ³⁶ (3) Infections lead to an increase in myeloid cells, especially monocytes and granulocytes. ^37,38 Our study found that KEGG pathways linked to infections like salmonella, E. coli, leishmaniasis, and others were enriched in CMML patients. We hypothesize that the TET2 mutation, susceptibility to co-infections, B-cell clearance after CD19 CAR-T therapy, and monocyte/macrophage activation by CRS collectively led to secondary CMML in this patient. Hematological patients receiving CAR-T therapy must carefully manage CRS and infections to reduce the risk of secondary tumors. Further research is needed to understand how CAR-T therapy might cause secondary CMML.

We analyzed immune cell changes in secondary CMML versus primary CMML and healthy controls, finding increased monocytes/macrophages (types 1, 2, 4, and 7) with upregulated interferon-inducible, pro-inflammatory, and "don't eat me" genes. This led to more exhausted T cells. ^35,39 The pro-inflammatory gene S100 family expression is closely associated with tumor progression and poor prognosis. ⁴⁰ The pro-inflammatory gene FCGR3A⁺ inflammatory monocytes/macrophages are thought to be involved in a variety of inflammation-related diseases in humans. ^41–44 Consistent with previous studies, FCGR3A⁺monocytes were found to express high levels of CX3CR1, which performs pro-inflammatory functions. ^45–48 The most well-described innate immune checkpoints are the “don’t eat me” signals, including the CD47/ SIRPα, PD-1/PD-L1 axis, CD24/SIGLEC-10 axis, and MHC-I/LILRB1/2 axis. ^49,50 We discovered that FCGR3A⁺ inflammatory monocytes/macrophages in secondary CMML highly express SIGLEC10 and LILRB2, indicating inhibited tumor phagocytosis. This underscores the importance of the immune microenvironment in secondary CMML. Our studies confirm that monocytes/macrophages promote tumors and induce immunosuppression, increasing dysfunctional T/NK cells in secondary CMML bone marrow. Reprogramming these cells to inhibit tumors could enhance treatment for secondary CMML. Additional experimental verification is necessary to substantiate our findings. Furthermore, conducting clinical trials would be valuable to validate this hypothesis.

The analysis of cell-cell communication in secondary CMML show the expression of pro-inflammatory cytokines and inflammation-related signaling pathways (IL1, CXCL, TNF, IFN-II, IL16, etc.), as compared to primary CMML. Previous studies have indicated that IL-1 signaling promotes malignant monocyte clones in CMML. ⁵¹ Anca Franzini et al. found that CMML monocytes exhibit a proinflammatory transcriptional signature (upregulation of TNF and IL-6 and − 17 signaling), contributing to malignant expansion and increased cardiovascular risk. ³³ Sandrine Niyongere et al. demonstrated that increased cytokine levels in IL-8, IP-10, IL-1RA, TNF-α, IL-6, MCP-1/CCL2, HGF, M-CSF, VEGF, IL-4, and IL-2RA can be observed in CMML. ⁵² In the present study, we found that the upregulation of CXCL, IFN-II, and IL16 on monocytes/macrophages may also play a significant role in the development of secondary CMML. Chemokines cause leukocytes to migrate to the site of inflammation, creating a localized inflammatory microenvironment. ^53,54 IFN-γ activates macrophages and promotes the Th1 response via IFNGR1 and IFNGR2 receptors, but prolonged signaling leads to immunosuppression.^35,55,56 IL16 induces a pro-inflammatory state in monocytes/macrophages and sustains inflammation.⁵⁷ Monocytes/macrophages likely have a complex role in regulating immune responses and inflammation within the BM microenvironment of secondary CMML. Additional research is required to understand the interactions between different cell types in both primary and secondary CMML.

HMAs are the only approved therapy for CMML. To improve response rates and survival, Venetoclax has been added, with conflicting being reported results in some studies. ^{16–18,20,58} However, there have not yet been any reports on the effect of Azacitidine in combination with Venetoclax on the BM immune microenvironment of patients with secondary CMML. Our study shows that treatment with Azacitidine and Venetoclax in secondary CMML increases the T/NK cell ratio and enhances their cytotoxic effects, while significantly reducing myelomonocytic cells, indicating treatment efficacy. Cell–cell communication analysis demonstrated that treatment with Azacitidine and Venetoclax significantly boosts the expression of apoptosis-related genes (THY1, ¹⁹ TRAIL, ²⁰ and APRIL ²¹), indicating that this combination may directly promote the apoptosis of myelomonocytic cells, thereby reducing their numbers. Previous studies have demonstrated that CD40 and CD23 can be expressed in myeloid cells and are regarded as T/NK cell activation molecules. ^22–24,59 This study found increased CD40 and CD23 expression in monocytes/macrophages, suggesting a role in T/NK cell immunomodulation. These cellular interactions may influence immune response and inflammation in secondary CMML post-treatment. Further research is needed to understand Azacitidine and Venetoclax's mechanisms in primary CMML patients.

In summary, we presented a high-resolution transcriptome map of primary and secondary CMML and deciphered the impact of Azacitidine in combination with Venetoclax on the immune microenvironment of secondary CMML. These findings will provide further new insights into CMML development and potential treatment mechanisms. This study has limitations, as it only evaluated one rare case of secondary CMML. We examined changes in the BM immune microenvironment before and after treatment and compared it with a primary CMML case. Our findings showed significantly higher monocyte/macrophage levels in both patients, suggesting targeted macrophage therapy could be effective for primary and secondary CMML. Future research should monitor BM microenvironment changes in primary CMML patients before and after treatment, explore targeted macrophage therapy strategies, and validate them through clinical trials. Such efforts aim to provide novel therapeutic options for CMML patients.

Acknowledgments

This work was supported by Natural Science Foundation of China (82270149, 82170211, 32100698, 82270141, 82370144, 82370219,82000163,82200190), Henan Province Medical Science and Technology Research Project (SBGJ202101007, LHGJ20220305, LHGJ20220301, LHGJ20200366, SBGJ202102146, SBGJ202102063), the Natural Science Foundation of Henan Province (222300420068, 242300421080, 222300420333, 222300420567). Key Research Projects of Henan Higher Education Institutions (222102310204). International Science and Technology Cooperation Program of Henan Provincial Science and Technology Department (232102521027) and Leading talent project of Henan Province (LJRC2023004). Funding for Scientific Research and Innovation Team of The First Affiliated Hospital of Zhengzhou University.

Conflict of interest

The authors declare no conflict of interest.

Author Contributions

WL, LX, and HS drafted the manuscript. XL, and HH designed and performed experiments. JS, XM and LG performed the ScRNA-seq analysis. YL analyzed the Flowcytometry data. MG, MS, HC, DZ, NS, WC, ZB, and YS read and edited the manuscript. WL, LX, and HS designed and supervised the study and edited the manuscript. All authors approved the final manuscript.

Data availability statement

All data generated in the study are included in the present article and supplementary data. The original 10x genomics transcriptomic sequencing data has been uploaded to NODE database (https://www.biosino.org/node), Accession Number: OEP005521.

Ethics Statement

This study was reviewed and approved by the First Affiliated Hospital of Zhengzhou University (2021-KY-0575-002).

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Decoding the Immune Microenvironment of Secondary Chronic Myelomonocytic Leukemia due to DLBCL with CD19 CAR-T Failure by Single-cell RNA-seq

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Abstract

Figures

1. Introduction

2. Materials and Methods

3.Results

3.1. Morphological and immunophenotypic differences between primary and secondary CMML

3.2. Bone marrow immune microenvironment features of primary CMML, and secondary CMML before treatment

3.3. Increased infection-mediated phagocytosis, chemotaxis and immune responses of monocytes/macrophages in secondary CMML

3.4. Transcriptomic differences of the monocytes/macrophages between primary CMML and secondary CMML

3.5. Decreased cytotoxicity of T/NK cells in patients with secondary CMML before treatment

3.6. Transcriptomic differences of T/NK cells between the primary CMML and secondary CMML

3.7. The potential indirect mechanisms of secondary CMML via cell–cell communication analysis

3.8. The remodeled BM immune cells in secondary CMML post Azacitidine plus Venecla treatment

3.9. The effect on monocytes/macrophages of Azacitidine plus Venecla treatment in secondary CMML

3.10. Sub-clustering of T/NK lineage in secondary CMML before and after Azacitidine plus Venecla treatment

4. Discussion

Declarations

References

Supplementary Files

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