CCRL2 affects the sensitivity of myelodysplastic syndrome and secondary acute myeloid leukemia cells to azacitidine

Abstract

Better understanding of the biology of resistance to DNA methyltransferase (DNMT) inhibitors is required to identify therapies that can improve their efficacy for patients with high-risk myelodysplastic syndrome (MDS). CCRL2 is an atypical chemokine receptor that is upregulated in CD34+ cells from MDS patients and induces proliferation of MDS and secondary acute myeloid leukemia (sAML) cells. In this study, we evaluated any role that CCRL2 may have in the regulation of pathways associated with poor response or resistance to DNMT inhibitors. We found that CCRL2 knockdown in TF-1 cells downregulated DNA methylation and PRC2 activity pathways and increased DNMT suppression by azacitidine in MDS/sAML cell lines (MDS92, MDS-L and TF-1). Consistently, CCRL2 deletion increased the sensitivity of these cells to azacitidine in vitro and the efficacy of azacitidine in an MDS-L xenograft model. Furthermore, CCRL2 overexpression in MDS-L and TF-1 cells decreased their sensitivity to azacitidine. Finally, CCRL2 levels were higher in CD34+ cells from MDS and MDS/myeloproliferative neoplasm patients with poor response to DNMT inhibitors. In conclusion, we demonstrated that CCRL2 modulates epigenetic regulatory pathways, particularly DNMT levels, and affects the sensitivity of MDS/sAML cells to azacitidine. These results support CCRL2 targeting as having therapeutic potential in MDS/sAML.

Figure 1.

CCRL2 knockdown downregulates pathways associated with DNA methylation and PRC2 activity. (A) RNA sequencing and gene set enrichment analysis in RNA collected from TF-1 cells transduced with shControl or shCCRL2 (sh1) lentiviruses and selected with puromycin demonstrated a number of oncogenic pathways downregulated by CCRL2 knockdown (KD). Among the topdownregulated pathways were pathways associated with DNA methylation and PRC2 activity. (B) Enrichment plots showing the suppression of PRC2 methylation activity and DNA methylation pathways by CCRL2 KD. (C) Western blot showing that CCRL2 KD suppressed DNMT3A and DNMT3B in TF-1 cells and all the DNMT in MDS-L cells. CCRL2 KD also increased p27 levels, decreased RB1 phosphorylation, and suppressed the protein levels of the PRCA component SUZ12, and the TFRC targets LIN28B and GNAQ in both TF-1 and MDS-L cells. CCRL2 KD decreased the nuclear levels of E2F1 in both TF-1 and MDS-L cells. (D) Box plot of normalized expression of selected genes encoding DNA methyltransferases (DNMT) and PRC2 components between CCRL2 knockout cells and controls. NES: normalized enrichment score; FDR: false discovery rate.

Figure 2.

CCRL2 knockdown increases DNMT protein suppression by azacitidine. (A-C) MDS92, MDS-L and TF-1 cells were treated with 0.5 mM azacitidine for 24 h following transduction with shControl or shCCRL2 (sh1) lentiviruses and selection with puromycin. Quantitative data and a representative western blot are shown. (A) The combination of CCRL2 knockdown (KD) and azacitidine treatment had a more prominent effect on the suppression of DNMT1 and DNTM3B levels in MDS92 cells compared to azacitidine (P=0.014 for DNMT1 and P<0.001 for DNMT3B) and shCCRL2 (P<0.001 for DNMT1 and P=0.009 for DNMT3B) separately. (B) The combination of CCRL2 KD and azacitidine treatment had a more prominent effect on the suppression of DNMT1 and DNTM3B levels in MDS-L cells compared to azacitidine (P<0.001 for both DNMT1 and DNMT3B) and shCCRL2 (P=0.008 for DNMT1 and P=0.001 for DNMT3B). (C). The combination of CCRL2 KD and azacitidine treatment had a more prominent effect on the suppression of DNMT1 and DNTM3B levels in MDS92 cells compared to azacitidine (P<0.001 for both DNMT3A and DNMT3B) and shCCRL2 (P=0.016 for DNMT3A and P=0.047 for DNMT3B).

Figure 3.

CCRL2 knockdown increases the clonogenicity inhibition and apoptotic effects of azacitidine. (A) CCRL2 knockdown (KD) by two different lentiviruses (sh1 and sh2) decreased the azacitidine half maximal inhibitory concentration (IC₅₀) for the clonogenicity of MDS92 cells (P<0.001 for both sh1 and sh2). (B) CCRL2 KD by two different lentiviruses (sh1 and sh2) decreased the azacitidine IC₅₀ for the clonogenicity of MDS-L cells (P<0.001 for both sh1 and sh2). (C) CCRL2 KD by two different lentiviruses (sh1 and sh2) decreased the azacitidine IC₅₀ for the clonogenicity of TF-1 cells (P<0.001 for both sh1 and sh2). (D) CCRL2 KD in MDS92 cells increased the percentage of early apoptotic and necrotic cells under treatment with 0.5 mM (P<0.001 with sh1 and P=0.005 with sh2) and 1 mM (P<0.001 with sh1 and P=0.007 with sh2) of azacitidine (n=3). (E) CCRL2 KD in MDS-L cells increased the percentage of early apoptotic and necrotic cells under treatment with 0.5 mM (P=0.028 with sh1 and P=0.003 with sh2) and 1 mM (P=0.003 with sh1 and P=0.035 with sh2) of azacitidine (n=3).

Figure 4.

CCRL2 knockdown increases the differentiation effect of azacitidine. (A) Treatment of MDS92 cells transduced with shCCRL2 (sh1 or sh2) lentiviruses with 0.5 and 1 μΜ azacitidine (Aza) led to a more prominent increase of CD11b (Aza 0.5: P<0.001 for both sh1 and sh2; Aza 1: P=0.001 for sh1 and P<0.001 for sh2), CD14 (Aza 0.5: P<0.001 for both sh1 and sh2; Aza 1: P<0.001 for both sh1 and sh2) and CD16 (Aza 0.5: P<0.001 for both sh1 and sh2; Aza 1: P<0.001 for both sh1 and sh2) expression compared to treatment of cells transduced with shControl lentivirus. Representative flow cytometry graph showing that CCRL2 KD increased the upregulation of CD16 on the surface of MDS92 cells caused by 1 mM azacitidine. (B) Treatment of MDS-L cells transduced with shCCRL2 (sh1 or sh2) lentiviruses with 0.5 and 1 mM azacitidine led to a more prominent increase of CD11b (Aza 0.5: P<0.001 for both sh1 and sh2; Aza 1: P<0.001 for sh1 and P<0.001 for sh2), CD14 (Aza 0.5: P=0.001 for both sh1 and sh2; Aza 1: P<0.001 for both sh1 and sh2) and CD16 (Aza 0.5: P<0.001 for both sh1 and sh2; Aza 1: P<0.001 for both sh1 and sh2) expression compared to treatment of cells transduced with shControl lentivirus. Representative flow cytometry graph showing that CCRL2 KD increased the upregulation of CD16 on the surface of MDS-L cells caused by 1 mM azacitidine. (C) Treatment of TF-1 cells transduced with shCCRL2 (sh1 or sh2) lentiviruses with 0.5 and 1 mM azacitidine led to a more prominent increase of CD41 (Aza 1: P=0.037 for sh2), CD61 (Aza 0.5: P<0.001 for sh1 and P=0.001 for sh2, Aza 1: P<0.001 for sh1 and P=0.005 for sh2), CD71 (Aza 0.5: P<0.001 for sh1, P=0.002 for sh2; Aza 1: P<0.001 for sh1, P=0.002 for sh2), CD235a (Aza 0.5: P=0.002 for sh1 and P=0.007 for sh2; Aza 1: P=0.010 for sh1 and P=0.030 for sh2) expression compared to treatment of cells transduced with shControl lentivirus. Representative flow cytometry graphs showing that CCRL2 KD increased the upregulation of CD71 and CD235a on the surface of TF-1 cells caused by 1 mM azacitidine (n=3).

Figure 5.

Doxycycline-induced CCRL2 overexpression increases the resistance of MDS-L and TF-1 cells to azacitidine. (A) Doxycycline-induced CCRL2 overexpression in MDS-L cells transduced with pLV-Puro-TRE-CCRL2- and pLV-Hygro-CMV>tTS/rtTA-expressing lentiviruses and selected with puromycin and hygromycin treatment was confirmed by western blot and flow cytometry. (B) CCRL2 overexpression by treatment of MDS-L cells with 1 mg/mL doxycycline led to a less prominent suppression of clonogenicity by 0.5 mM azacitidine (P=0.019) and increase of the azacitidine half maximal inhibitory concentration (IC₅₀) (P=0.003). (C) CCRL2 overexpression by treatment of MDS-L cells with 1 mg/mL doxycycline led to a small decrease of the percentage of apoptotic cells under treatment with 0.5 mM (P=0.010) and 1 mM azacitidine (P=0.001). (D) Doxycycline-induced CCRL2 overexpression in TF-1 cells with CRISPR-Cas9 CCRL2 deletion, transduced with pLV-Puro-TRE-CCRL2-and pLV-Hygro-CMV>tTS/rtTA-expressing lentiviruses and selected with puromycin and hygromycin treatment, was confirmed by western blot and flow cytometry. (E) CCRL2 overexpression by treatment of TF-1 cells with 1 mg/mL doxycycline led to a higher clonogenic capacity (P<0.001) in cells treated with dimethylsulfoxide and a less prominent suppression of clonogenicity by 0.5 mM (P=0.001) and 1 mM azacitidine (P=0.002). This led to a less prominent decrease of the relative number of colonies in cells treated with 1 mM azacitidine (P=0.044) and an increased azacitidine IC₅₀ (P=0.011). Doxy: doxycycline; Aza: azacitidine; DMSO: dimethylsulfoxide.

Figure 6.

Combination treatment with CCRL2 knockdown and azacitidine leads to MDS-L growth suppression in NSGS mice. (A) Monitoring the bioluminescence signal showed that NSGS mice engrafted with MDS-L cells transduced with shCCRL2 lentivirus treated with intravenous azacitidine (2.5 mg/kg/day every 5 days for 5 doses) showed the smallest disease growth compared to mice engrafted with MDS-L cells transduced with shControl lentivirus and treated with dimethylsulfoxide (DMSO) or azacitidine and mice engrafted with MDS-L cells transduced with shCCRL2 lentivirus and treated with DMSO. (B) Mice engrafted with MDS-L cells transduced with shCCRL2 and treated with azacitidine had lower disease burden in their bone marrow based on the percentage of human CD45 (hCD45+%) cells compared to mice engrafted with shControl transduced cells and treated with azacitidine (P=0.004) and mice engrafted with shCCRL2 transduced cells and treated with DMSO (P=0.008). AZA: azacitidine.

Figure 7.

CCRL2 expression in CD34⁺ cells is negatively correlated with response to DNMT inhibitors. (A) Genomic landscape of patients with myelodysplastic syndrome (MDS) and MDS/myeloproliferative neoplasms (MPN) included in the analysis sorted by CCRL2 levels with gender, disease subtype (MDS and MDS/MPN) and specific somatic mutations being indicated. MDS/MPN patients had higher levels of CCRL2 (P=0.033) and men had relatively higher levels compared to women (P=0.047). Patients who achieved complete or partial remission (CR or PR) are presented in blue, patients with stable disease (SD) are presented in orange and patients with progressive disease (PD) are presented in red. (B) Patients with CR/PR had lower CCRL2 levels in their CD34⁺ cells compared to patients with SD (P=0.029) or PD (P=0.002). (C) Kaplan-Meier analysis showed that CCRL2 protein levels above the median were associated with worse overall survival since diagnosis (P=0.039) compared to CCRL2 protein levels below the median.