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  • CXCL1, CXCL10 and CXCL12 Chemokines are Variously Expressed in Acute Myeloid Leukemia Patients Prior and Post Bone Marrow Transplantation

  • Dr. Hossein Khorramdelazad ,1,*


  • Introduction: The malignancy state of AML (acute myeloid leukemia) is characterized as the uncontrolled proliferation of myeloid leukocyte blasts within the bone marrow (BM) with arrested maturation processes. Similarly as, normal precursors, leukemic cells are also able to communicate with their surrounding hematopoietic microenvironment in several paths (Giles et al., 2002). AML is more frequent in elder adults, and is reported to be worsened with increasing age. The incidence of AML increases with age (a median age at its diagnosis is 67 years) (National Cancer Institute. Surveillance, 2009) . BMT is a promising therapy for acute leukemia which is widely used for therapy of relapsed AML patients. However, the benefits of BMT for patients with late BM relapse or multiple relapses have yet to be firmly clarified (Butturini et al., 1987; Moradabadi et al., 2019) . The CXC chemokine of stromal cell-derived factor-1 (SDF-1α) which in the latest nomenclature was defined as CXCL12 is described as a potent chemotactic factor for the human CD34 positive pluripotent stem cells (Aiuti et al., 1997). Despite the pro inflammatory chemokines, CXCL12 is constitutively produced and generated by several body organs including human BM (Yazdani et al., 2020; Ponomaryov, 2000). This chemokine plays fundamental parts in homeostatic events including control of leukocyte trafficking, retention of undifferentiated and maturing hematopoietic cells inside BM in both the normal and pathologic states, in addition to CXCR4 and CXCR7, as its related receptors (Leslie RD, 1999;42:3-14; Mousavi et al., 2019) . The disrupted CXCL12/CXCR4 and CXCL12/CXCR7 axes regulate anchorage leading to the release of cells into the circulation(Broxmeyer HE, 2001). It has been well evidenced that degradation of BM originated, CXCL12 by proteolytic enzymes, leading to the release of progenitor and mature cells from the BM towards the periphery in response to granulocyte colony-stimulating factor (G-CSF) -induced mobilization (Petit et al., 2002). Investigations revealed that some of the human CXCR4 expressing AML cells are chemo attracted toward a gradient of CXCL12 in a trans endothelial migration assay in vitro (Mohle et al., 1998; Dabiri et al., 2018). Since CXCL12 is constitutively expressed by BM stromal cells, it could be speculated that this chemokine contributes to the migration and survival of leukemic blasts during the pathogenesis of acute leukemia (Yoshie et al., 2001). Besides playing a pivotal part in migration, there also exist reports indicating that CXCL12 might be involved in the pathogenesis of leukemia (Nishii et al., 1999). Although, few studies addressed a role for CXCL12 in AML, involvement of CXCL1 and CXCL10 in the pathogenesis of this malignancy yet to be elucidated. Therefore, we designed the current investigation to explore whether if the expression of CXCL1, CXCL10 and CXCL12 in parallel with their cognate receptors is altered before and following BMT in AML patients. Chemokines and their receptors are important in the recruitment of leukocytes to the rejecting allograft. These mediators and their corresponding receptors are specifically expressed in human kidney, heart and lung allografts. Thus, we also aimed to present investigation to detect the CXC chemokines in bone marrow transplanted AML patients and chemokines in relation to clinical status, outcome therapeutic and severity of the disease in BMT received patients.
  • Methods: The research involving human participants and all of the participants had Informed consent. This project was performed during 2015-2017 and 46 AML patients (23 AML- M1 and 23 AML_M3) were enrolled in the study at the Aliebne-Abitaleb Hospital in Rafsanjan, located in South-East of Iran. The type of AML (either M1 or M3) was diagnosed by pathological studies based on the ratio of cell types observed within the BM. According to the aim of the study, the serum CXCL1, CXCL10 and CXCL12 levels were examined before and following BMT. We have also explored the expression of CXCR1, CXCR3 and CXCR4 in peripheral blood mononuclear cells (PBMCs) of patients. The occurrence of AML was diagnosed by an expert clinical hemato-oncologist, of course, based on clinical and Para clinical parameters such as BM and peripheral blood smear studies along with chemical staining and also clinical features of the patients. All of the prior BMT samples were collected further extensive chemotherapy. To collect post BMT samples, patients received entire and complete BMT. All of samples were also obtained from patients who have not shown evidence of either acute, sub-acute or chronic graft rejection. Patients were recruited if exhibited no evidence for infection, injury or inflammation. Having injury, infection, autoimmunity and known disorder which may change the chemokine expression were considered as excluding criteria. All of the clinical samples were collected in the morning following ambulatory visits and 5 mL of blood was taken from the patient. Serum samples were kept at -20°C for further analysis in the Molecular Medicine Research Center, Rafsanjan University of Medical Sciences, Rafsanjan-Iran. We selected healthy individuals from the Kerman population as the control group and were then matched with AML patients about demography, including age and sex status. All of the participants have filled a written consent out form and the study program was approved by the Rafsanjan University of Medical Science regional ethical committee. Assessment of chemokines by ELISA The CXCL1, CXCL10 and CXCL12 serum levels were measured by ELISA (R&D systems, UK). All of assays were conducted according to manufacturer’s guidelines. RNA isolation and QRT-PCR The PBMCs were harvested using density gradient separation (Ficoll–Hypaque; NyCoMed, Oslo, Norway; specific density 1.077) from the Peripheral blood samples of patients and then, the total RNA was isolated from PBMCs employing trizol in accordance with the manufacturer’s instructions (Invitrogen, USA). cDNA was synthesized from total RNA (Thermo Scientific, USA) all of assays were conducted according to manufacturer’s guidelines. The reference gene of GAPDH was selected based on a uniform expression in all samples. QR-PCR was performed for mRNA detection of chemokine receptors CXCR1, CXCR3, CXCR4, by applying Power SYBR Green PCR Master Mix (Amplicone) in a Rotor-Gene Q system the following conditions: 95°C for 15 min and 40 cycles at 95°C for 15 s and 60°C for 1 min. The specific Primer sequences for chemokines receptors are listed in Table 1. Statistical analysis Statistical analysis of the data was assessed by χ2, T-Test and ANOVA using SPSS software version 18 with power test of 90%. The difference regarded significant, if the P value was less than 0.05.
  • Results: Patients To investigate the fundamental role played by the CXC chemokines, including CXCL1, CXCL10 and CXCL12 in AML patients prior and post BMT, the present study was carried out on 46 AML patients (23 AML-M1 and 23 AML-M3). Our results showed that the mean age in male patients was 24(64.86%). Our findings also demonstrated that the mean age in female patients was 13(35.1%). 5(3.1%) of patients had familial history of cancer (Table 2). Level of Serum Chemokines Further data analysis, our data indicated that the expression of CXCL1, CXCL10 and CXCL12 was not associated with the gender of AML patients. We did not also find a relationship between the age and expression of chemokines in AML patients. We did find a significant difference in the CXCL1 level between M1 subtype and M3 subtype (P<0.01) (Table 3). We have observed a remarkable difference between AML subtypes regarding CXCL10 expression, M1 and M3 subtypes (P<0.01) (Table 3).
  • Conclusion: This study investigated the serum levels of CXCL1, CXCL10, and CXCL12 chemokines, along with the mRNA expression of their cognate receptors (CXCR1, CXCR3, and CXCR4), in patients with acute myeloid leukemia (AML) before and after bone marrow transplantation (BMT). The findings reveal distinct expression patterns among these chemokines, underscoring their potential roles in the pathogenesis of AML and in the context of BMT.
  • Keywords: AML- leukemia- CXC chemokine- CXC chemokine receptor- BMT

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