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

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

RAR-Alpha Targeting Compounds Overcome Bone Marrow (BM) Stromal Protection of AML By CYP26

Abstract All-trans retinoic acid (ATRA) causes terminal differentiation and apoptosis of non-APL AML cells in vitro, but has not proven clinically effective. We have recently shown that BM stroma expressing CYP26 inactivates ATRA and protects leukemia cells from differentiation (Su M et al 2015). ATRA signals through retinoic acid receptors (RARs) α, β and γ and induces not only terminal differentiation of AML, but also upregulation of CYP26, potentially forming an even more protective niche for leukemia. In an effort to decouple these two biological effects and improve differentiation therapy for AML, we investigated the relative contribution of RARα and RARγ to differentiation and CYP26 upregulation. ATRA (pan-RAR agonist), AM80 (strong RARα agonist, weak RARγ), and IRX5183 (RARα-specific agonist; previously known as NRX 195183, Io Therapeutics, Inc.) all induced significant differentiation of NB4 APL cells (i.e., upregulation of myeloid differentiation antigen CD11b and decreased clonogenic activity) at 0.1 μM. In contrast, the RARγ-specific agonist CD437 (0.1 μM) produced no evidence of differentiation. To determine the impact of RARα and RARγ signaling on stromal CYP26B1 levels, we treated murine OP9 BM stroma cells in low serum conditions with the aforementioned agents. ATRA induced a 40.5±30.1 fold upregulation of CYP26B1 at 24h (p=0.02 compared to control) and 24.7±20.4 fold upregulation at 48 hours (p=0.03). CD437 showed progressive upregulation of CYP26B1 (14.4±5.2 fold at 24h and 27.7±8.8 fold at 48h, p&lt;0.01 for both). In contrast, the RARα-specific IRX5183 had only modest effects on CYP26B1 levels (2.9±0.9 fold at 24h and 2.7±0.2 fold at 48h, p&lt;0.01 for both). AM80 showed the highest up-regulation of CYP26B1 levels both at 24h and 48h of treatment (29.1±7.9 and 77±24.4, p&lt;0.01 for both). Since RARγ activation appeared to be dispensable for differentiation, we tested if the two RARα active agents, AM80 and IRX5183, could bypass stromal-mediated protection against ATRA-induced differentiation, as both of these compounds have also been reported to be resistant to CYP26 mediated degradation. Several AML cell lines were cultured in the presence or absence of OP9 BM stroma and treated with ATRA, AM80 or IRX5183 at concentrations of 0.01 to 0.1 μM. We have previously shown that ATRA can induce differentiation of most AML cell lines in stroma-free conditions, but it is inactive in the presence of BM stroma; further, its effect can be restored by inhibition of stromal CYP26 (Su M et al. 2015). Whereas stroma blocked upregulation of CD11b and inhibition of clonogenicity of NB4 cells by ATRA, both AM80 and IRX5183 showed similar activity in the presence or absence of stroma (e.g. AM80 resulted in a fold change CD11b from control of 9.8±5.3 off stroma vs 11.8±8.4 on stroma, p=NS). Stroma also blocked ATRA's activity against NPM1-mutated OCI-AML3 cells (clonogenic recovery from control 6.4±3.3% vs 61.4±12.2% on stroma, p=0.002), but AM80 and IRX5183 were active both in the presence and absence of stroma (e.g. IRX5183 resulted in clonogenic recovery from control of 7.3±2.5% vs 11.2±2.8% on stroma, p=NS). Similar effects were observed using Kasumi-1 core binding factor AML cells. In conclusion, we found that RARα and γ have distinct effects on niche vs AML cells. Stimulation of RARγ does not induce differentiation of AML cells, but leads to the upregulation of stromal CYP26B1, and thus enhanced resistance to ATRA. In contrast, CYP26 resistant, RARα active synthetic retinoids AM80 and IRX5183 are able to differentiate and eliminate AML cells even in the presence of BM stroma regardless of induction of CYP26 expression. We are currently exploring a phase I/II clinical trial using IRX5183 in non-APL AML. Disclosures Chandraratna: Io Therapeutics, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: President. </jats:sec

Abstract 5435: CCRL2 affects the sensitivity of MDS and secondary AML to azacitidine

Abstract INTRODUCTION: We recently found that the atypical chemokine receptor, CCRL2 promotes the growth of MDS and secondary AML (sAML) while CCRL2 knockdown inhibits their growth. The aim of the current study is to investigate if CCRL2 regulates pathways associated with the development of resistance to hypomethylating agents (HMA), commonly used drugs in MDS and occasionally sAML. MATERIALS AND METHODS: We used lentivirus-mediated transduction of MDS92, MDS-L and TF-1 MDS/sAML cells to suppress CCRL2 expression. Two different shRNA constructs were used. We performed RNA sequencing and gene-set enrichment analysis of CCRL2 knocked down (KD) and wild-type (WT) TF-1 cells. We measured DNA methyl-transferases’ expression by western blot, CD11b and CD71 expression was measured in MDS and sAML cells to assess cell differentiation. Apoptosis was measured by Annexin V/PI staining, and clonogenicity by methylcellulose assays. CD34+ cells were sorted from bone marrow aspirates of MDS patients before the initiation of treatment with HMA by using magnetic beads and measurement of CCRL2 was performed by flow cytometry. Response to HMA in MDS patients was assessed by 6 months of treatment based on the International Working Group response criteria. RESULTS: CCRL2 KD cells demonstrated suppression of pathways associated with PRC2 complex activity, histone modification, and DNA methylation. CCRL2 KD also lead to a more prominent degradation of DNMT1, DNMT3A and DNTM3B under azacitidine treatment. CCRL2 increased apoptosis in response to 0.5 and 1 μM azacitidine (P&amp;lt;0.010) and MDS-L cells (P&amp;lt;0.010 with both sh1 and sh2). Similarly, CCRL2 knockdown increased morphologic differentiation with both 0.5 and 1 μΜ azacitidine (P&amp;lt;0.010) as well as increased the clonogenic inhibition caused by azacitidine (P&amp;lt;0.05). In order to analyze the effect CCRL2 clinically, we analyzed CCRL2 expression in CD34+ cells from patients undergoing HMA treatment. Non-responders to HMA (progressive disease) express higher levels of CCRL2 compared to CD34+ cells from responders (complete remission, partial remission or stable disease) (P=0.020). DISCUSSION: Our analysis suggests that CCRL2 regulates the expression of genes associated with induction of PRC2-mediated histone modification and DNA methylation in sAML cells. CCRL2 suppression also increased the sensitivity of MDS and sAML cells to azacitidine. In addition, increased CCRL2 expression in MDS cells is associated with worse response to HMA. These data suggest that targeting CCRL2 has therapeutic potential in MDS and sAML. Citation Format: Theodoros Karantanos, Patric Teodorescu, Brandy Perkins, Marios Arvanitis, Ilias Christodolou, Christopher Esteb, W. Brian Dalton, Tania Jain, Amy E. DeZern, Lukasz P. Gondek, Mark J. Levis, Gabriel Ghiaur, Richard J. Jones. CCRL2 affects the sensitivity of MDS and secondary AML to azacitidine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5435.</jats:p

Silencing of the DNA damage repair regulator PPP1R15A sensitizes acute myeloid leukemia cells to chemotherapy

Acute Myeloid Leukemia (AML) is a life-threatening disease whose induction treatment consists of combination chemotherapy with Idarubicin and Cytarabine for fit patients. Treatment failures are frequent, urging the need for novel treatments for this disease. The DNA Damage Response Mechanism (DDR) comprises numerous molecules and pathways intended to arrest the cell cycle until DNA damage is repaired or else drive the cell to apoptosis. AML-derived cell lines after treatment with Idarubicin and Cytarabine were used for studying the expression profile of 84 DDR genes, through PCR arrays. Utilizing de novo AML patient and control samples we studied the expression of PPP1R15A, CDKN1A, GADD45A, GADD45G, and EXO1. Next, we performed PPP1R15A silencing in AML cell lines in two separate experiments using siRNA and CRISPR-cas9, respectively. Our findings highlight that DDR regulators demonstrate increased expression in patients with high cytogenetic risk possibly reflecting increased genotoxic stress. Especially, PPP1R15A is mainly involved in the recovery of the cells from stress and it was the only DDR gene upregulated in AML patients. The PPP1R15A silencing resulted in decreased viability of Idarubicin and Cytarabine-treated cell lines, in contrast to untreated cells. These findings shed light on new strategies to enhance chemotherapy efficacy and demonstrate that PPP1R15A is an important DDR regulator in AML and its downregulation might be a safe and effective way to increase sensitivity to chemotherapy in this disease. © The Author(s) 2024

The role of the atypical chemokine receptor CCRL2 in myelodysplastic syndrome and secondary acute myeloid leukemia

ABSTRACTThe identification of new molecular pathways supporting the growth of myelodysplastic syndrome (MDS) stem and progenitor cells is needed to understand clinical variation and develop targeted therapies. Within myeloid malignancies, men have worse outcomes compared to women, suggesting male sex hormone driven effects in malignant hematopoiesis. The androgen receptor promotes the expression of five granulocyte-colony factor receptor regulated genes. Among them, CCRL2 encodes an atypical chemokine receptor that regulates cytokine signaling in differentiated granulocytes but its role in myeloid malignancies is unknown. Our study revealed that CCRL2 is upregulated in stem and progenitor cells from patients with MDS and secondary acute leukemia. CCRL2 knockdown suppressed the growth and clonogenicity of MDS92 and MDS-L cells in vitro and in vivo. Moreover, CCRL2 knockdown significantly suppressed the phosphorylation of JAK2, STAT3, and STAT5 in MDS cells. CCRL2 co-precipitated with JAK2 and its suppression decreased the interaction of JAK2 with STAT proteins. Cell lines expressing JAK2V617F showed less effect of CCRL2 knockdown on growth and clonogenicity compared to those expressing wild type. However, the selective JAK2 inhibitor fedratinib potentiated the effects of CCRL2 knockdown in MDS and leukemia cells expressing both wild type JAK2 and JAK2V617F. In conclusion, our results implicate CCRL2 as a mediator of MDS and secondary acute leukemia cell growth, in part through JAK2/STAT signaling.</jats:p

The role of the atypical chemokine receptor CCRL2 in myelodysplastic syndrome and secondary acute myeloid leukemia

The identification of new pathways supporting the myelodysplastic syndrome (MDS) primitive cells growth is required to develop targeted therapies. Within myeloid malignancies, men have worse outcomes than women, suggesting male sex hormone–driven effects in malignant hematopoiesis. Androgen receptor promotes the expression of five granulocyte colony-stimulating factor receptor–regulated genes. Among them, CCRL2 encodes an atypical chemokine receptor regulating cytokine signaling in granulocytes, but its role in myeloid malignancies is unknown. Our study revealed that CCRL2 is up-regulated in primitive cells from patients with MDS and secondary acute myeloid leukemia (sAML). CCRL2 knockdown suppressed MDS92 and MDS-L cell growth and clonogenicity in vitro and in vivo and decreased JAK2/STAT3/STAT5 phosphorylation. CCRL2 coprecipitated with JAK2 and potentiated JAK2-STAT interaction. Erythroleukemia cells expressing JAK2V617F showed less effect of CCRL2 knockdown, whereas fedratinib potentiated the CCRL2 knockdown effect. Conclusively, our results implicate CCRL2 as an MDS/sAML cell growth mediator, partially through JAK2/STAT signaling. </jats:p