Knockout of LASP1 in CXCR4 expressing CML cells promotes cell persistence, proliferation and TKI resistance

Abstract

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the constitutively active BCR-ABL tyrosine kinase. The LIM and SH3 domain protein 1 (LASP1) has recently been identified as a novel BCR-ABL substrate and is associated with proliferation, migration, tumorigenesis and chemoresistance in several cancers. Furthermore, LASP1 was shown to bind to the chemokine receptor 4 (CXCR4), thought to be involved in mechanisms of relapse. In order to identify potential LASP1-mediated pathways and related factors that may help to further eradicate minimal residual disease (MRD), the effect of LASP1 on processes involved in progression and maintenance of CML was investigated. The present data indicate that not only overexpression of CXCR4, but also knockout of LASP1 contributes to proliferation, reduced apoptosis and migration as well as increased adhesive potential of K562 CML cells. Furthermore, LASP1 depletion in K562 CML cells leads to decreased cytokine release and reduced NK cell-mediated cytotoxicity towards CML cells. Taken together, these results indicate that in CML, reduced levels of LASP1 alone and in combination with high CXCR4 expression may contribute to TKI resistance.

Keywords: BCR-ABL; CML; CXCR4; LASP1; nilotinib; precursor cells.

Figure 1

Validation of K562 cells with inactivated LASP1 and CXCR4 overexpression. A, Western blot analysis (10% gel) of LASP1 and CXCR4 in K562 cells after CRISPR/Cas9‐mediated LASP1 knockout and after lentiviral transduced CXCR4 expression of indicated cell lines. Relative expression of CXCR4 mRNA (B) and of LASP1 mRNA (C) in the generated cell lines was analysed by qRT‐PCR as described in the Materials and Methods section. Results represent the mean of three independent experiments ± SD. D, CXCR4 functionality in CXCR4 overexpressing K562 cells was validated by AKT1‐S473 phosphorylation after 25 nmol/L (200 ng/mL) CXCL12 stimulation in a time dependent manner by Western blot analysis (10% gel). β‐actin served as loading control. E, Flow cytometric analysis of CXCR4 surface expression (50 000 recorded events). CXCR4 cell surface expression in CXCR4 overexpressing cell lines exceeded non‐transduced cell lines. CXCL12, C‐X‐C motif chemokine 12; CXCR4, chemokine receptor 4; DMSO, dimethyl sulfoxide; LASP1, LIM and SH3 domain protein 1; SD, standard deviation

Figure 2

Effect of LASP1 knockout and CXCR4 overexpression on cell growth and viability. K562‐LASP1↑‐CXCR4↑, K562‐LASP1↑‐CXCR4↓, K562‐LASP1↓‐CXCR4↑ and K562‐LASP1↓‐CXCR4↓ cell lines were tested for viability using RealTime‐Glo™ MT Cell Viability Assay (Promega) according to the manufacturer's protocol in (A) DMSO control, (B) after stimulation with 12.5 nmol/L (100 ng/mL) CXCL12 and (C) after incubation with 60 nmol/L nilotinib. Results represent the mean of three independent experiments in triplicates ± SD. CXCL12, C‐X‐C motif chemokine 12; CXCR4, chemokine receptor 4; DMSO, dimethyl sulfoxide; LASP1, LIM and SH3 domain protein 1; SD, standard deviation

Figure 3

Influence of LASP1 knockout and CXCR4 overexpression in K562 cell lines on apoptosis, cell cycle and flow adhesion. Apoptosis (A and B) and cell cycle arrest (C and D) were measured cytometrically using annexin V/PI staining. LASP1 knockout was of advantage in CXCR4 overexpressing cells while co‐expression of both proteins increased susceptibility towards TKI treatment. Results represent the mean of three independent experiments ± SD. CXCR4, chemokine receptor 4; DMSO, dimethyl sulfoxide; LASP1, LIM and SH3 domain protein 1; PI, propidium iodide; SD, standard deviation; TKI, tyrosine kinase inhibitor

Figure 4

Importance of LASP1 and CXCR4 for migratory potential. (A) Schematic of a migration chamber and assay conditions: Fluorescent signal from actively migrating cells was detected from below without shine‐through artefacts. Relative migration towards gravitation (B), 10% FCS (C) and 12.5 nmol/L (100 ng/mL) CXCL12 (D). In the presence of CXCR4, knockout of LASP1 resulted in reduced migration. (E) Cytospin and Pappenheim staining of migrated cells. Cytospins were prepared as previously described.9 Results represent the mean of three independent experiments in duplicates ± SD. BSA, bovine serum albumin; CXCL12, C‐X‐C motif chemokine 12; CXCR4, chemokine receptor 4; FCS, foetal calf serum; LASP1, LIM and SH3 domain protein 1; SD, standard deviation

Figure 5

Effect of LASP1 on K562 cell adhesion. A, Cell adhesion was tested under flow conditions. Adherent cells are visible as dots, floating cells as lines. 1 square is equivalent to 1 mm². B, LASP1 knockout reinforced adhesion by trend. Results represent the mean of three independent experiments ± SD. CXCR4, chemokine receptor 4; HUVEC, human umbilical vein endothelial cells; LASP1, LIM and SH3 domain protein 1; SD, standard deviation

Figure 6

Effect of LASP1 and CXCR4 on K562 cytokine release and NK cell degranulation. Cytokine release of IL‐6 (A), IL‐8 (C) and MCP‐1 (E) into the conditional medium was measured using a bead‐based immunoassay. Relative mRNA expression of IL‐6 (B), IL‐8 (D) and MCP‐1 (F). G, Degranulation response, assessed by flow cytometric analysis of CD107a surface expression of NK‐92C after 4‐h co‐incubation with K562 cells. Results represent the mean of three independent experiments ± SD. CXCR4, chemokine receptor 4; DMSO, dimethyl sulfoxide; LASP1, LIM and SH3 domain protein 1; MCP‐1, monocyte chemoattractant protein‐1; SD, standard deviation

Figure 7

Negative effect of impaired LASP1 levels worsen patients' outcome: a case presentation. A 66‐year‐old woman presented 5 months after being diagnosed to suffer from CML in chronic phase in 2011 (157.4 Gpt/L white blood cells) at the university hospital in lymphatic blast crises with low LASP1 levels (blue line), despite initiated imatinib therapy. Cytarabin, vincristine and hydroxycarbamide (indicated as ) were administered unsuccessfully. As cytogenetics finally revealed the presence of Y253H, E255K and T315I mutations, ponatinib (dotted line) was initiated. Subsequently, the administered pre‐phase chemotherapy consisted of methotrexate, dexamethasone and cyclophosphamide (indicated as ) according to German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia.44 Induction therapy had to be interrupted due to clinical deterioration. Finally, the patient underwent conditioning therapy with treosulfan, fludarabine and antithymocyte globulin45 followed by allogenic peripheral blood stem cell transplantation of 4.1 × 10⁶ CD34+ cells/kg bodyweight (indicated as ▼) from an unrelated male, human leucocyte antigen allele matched 10/10 donor. After peripheral blood stem cell transplantation, BCR‐ABL levels declined, while LASP1 levels increased. Due to a renewed blast crisis (and concomitant lowered LASP1 levels), the patient died 83 d after SCT (indicated as ) despite further doses of cyclophosphamide (indicated as ). The provenience and preparation of blood samples have been described before.9 CML, chronic myeloid leukaemia; CT, chemotherapy; LASP1, LIM and SH3 domain protein 1; PBSCT, peripheral blood stem cell transplantation; TKI, tyrosine kinase inhibitor