Resumen de tesis. Inhibition of SHP1 and SHP2 as a molecular targeted therapy against myeloid leukaemias

Abstract

[EN]Haematopoiesis is a very relevant differentiation process in adult humans where a multipotent cell, the haematopoietic stem cell (HSC), generates a widely varied, fully differentiated progeny, with immune defence, nutrient exchange and volume homeostasis functions. The regulatory cues governing the biology of HSCs must be tightly regulated in order to ensure their own self-renewal, as well as the proper turnover of differentiated cells. These signals are provided by the surrounding environment, known as niche, integrated by both haematopoietic and non-haematopoietic cells. The disruption of this fine equilibrium by alteration of either the external signals or their intracellular transduction in haematopoietic stem and progenitor cells (HSPCs) leads to the development of haematologic malignancies, including leukaemia. Two important blood disorders affecting the myeloid lineage are acute and chronic myeloid leukaemia (AML and CML, respectively). They are especially recurrent among the elderly, with a median age at diagnosis of 65 years for CML and 70 for AML, a fact to be considered due to the increasing life expectancy in Western countries. AML is a highly heterogeneous and aggressive disease with poor prognosis and, in general, no significant therapeutic improvements beyond chemotherapy over the last four decades. An exception to this scenario is the treatment of acute promyelocytic leukaemia (APL), which is highly responsive to pro-differentiative therapy, consisting of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). Unlike AML, CML is highly homogeneous in terms of molecular biology, with the expression of the fusion oncokinase breakpoint cluster region-ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL) as the main pathogenic driver. In the early 2000s, the clinical use of tyrosine kinase inhibitors (TKI) targeting this protein revolutionised the management of CML due to great improvements in treatment response and survival rates. However, this disease remains challenging in particular cases. Despite its heterogeneous nature, AML displays a differentiation blockage as a hallmark. This feature, together with the example of the differentiation-based APL treatment, has prompted the development of an important line of research focusing on the molecular mechanisms governing cell differentiation during haematopoiesis. This knowledge would lead to a better understanding of the dysregulated processes leading to pathogenesis and their subsequent pharmacological targeting to treat the disease. In line with this, the present work sought to assess in detail the involvement of SRC homology 2 domain containing protein tyrosine phosphatases 1 (SHP1) and 2 (SHP2) in the differentiation of leukaemic cells and the potential of these molecules as pharmacological targets for AML. Herein, it was demonstrated the cooperative function of both phosphatases in phorbol ester-induced cell differentiation, with an enhanced differentiated phenotype of cells subjected to simultaneous downregulation of these proteins. In addition, the kinase SRC was identified as a downstream target of SHP2 in this process, which appeared to influence the extent of the differentiation stimulus triggered by phorbol 12-myristate-13-acetate (PMA). Besides, the role of both phosphatases on cell differentiation showed to be partially overlapping through the regulation of β-catenin protein levels. Based on this evidence, the chemical inhibitor of SHP1 and SHP2 NSC 87877 (NSC) was successfully tested to boost the differentiation-inducing effect of phorbol esters in the AML cell line HL-60. Moreover, this compound synergised with the phorbol ester 13-O-acetyl-12-deoxyphorbol or prostratin (PRS) to prevent proliferation of not only in HL-60 cells, but also additional cell lines used as AML models (NB-4, OCI-AML2 and THP-1). Most importantly, the anti-leukaemic activity of this combination was corroborated in vivo with a xenograft mouse model and in primary cells from AML patients ex vivo. On the other hand, the management of CML still requires further improvement, since the success of TKI relies on long-term administration to patients due to the existence of quiescent BCR-ABL independent leukaemic stem cells (LSCs). This is associated with intolerances in patients and great costs for national health systems. Moreover, selective pressure on leukaemic blasts can lead to the emergence of point mutations in BCR-ABL that confer acquired resistance to TKI. To overcome this issue, novel therapeutic approaches based on co-targeting BCR-ABL and other important contributors to CML pathogenesis are subject of intense research. Based on this idea and the involvement of SHP1 and SHP2 in the disease, NSC was tested in combination with TKI in different models of CML. This compound enhanced the effect of the TKI imatinib (IM) and nilotinib (NL) in different CML cell lines (K-562, KCL-22 and BV-173). Immunoblotting analyses allowed the observation of a strong decrease in β-catenin levels upon NSC treatment and a moderate downregulation of c-MYC induced by both IM and NSC. Further gene expression studies identified CCND1, CCND2, CDKN1C and TLE2 as targets of either one or both drugs that might mediate the anti-leukaemic activity of their combination. Additionally, NSC also displayed anti-proliferative potential in patient-derived induced pluripotent stem cells (iPSCs), intrinsically resistant to IM at moderate doses. Treatment of these cells with NSC exerted a dramatic cell cycle arrest concomitant with the downregulation of CTNNB1, encoding β-catenin, as well as CCND1 and CCND2. Additionally, some members of TLE family were modulated by this drug. In summary, the results described in the present work support a promising therapeutic potential of the chemical inhibitor NSC 87877 in different myeloid malignancies where SHP1 and SHP2 are deeply involved. Furthermore, some mechanistic insight on the molecules connected to these phosphatases in both disease biology and pharmacological mode of action of the inhibitor has been provided.