Functional characterization of a receptor protein associated with poor therapy response and relapse of acute myeloid leukemia

Most patients with acute myeloid leukemia (AML) initially respond to chemotherapy and achieve a complete remission. However, a large proportion of them eventually suffer relapse, a stage at which lasting therapeutic successes are rarely achieved. Relapse, resulting from the accumulation of malignant cells that resisted the primary therapy, therefore represents a central problem in the treatment of AML. To discover molecular mechanisms that may play a role in cellular therapy resistance and the ensuing disease recurrence, genes that are consistently deregulated between diagnosis and relapse of AML were sought through genome wide gene expression profiling. Among 463 genes resulting from this screen, several additional selection steps identified the calcitonin receptor-like (CALCRL) gene as a particularly interesting candidate. CALCRL codes for a G-protein coupled seven- transmembrane receptor that can be activated by either of two peptides, adrenomedullin (ADM) or calcitonin gene related peptide (CGRP), in a manner depending on the presence of specific coreceptors. Even though CALCRL and its peptide ligands have not previously been implicated in AML, they were reported to play a role in the pathogenesis of other malignant diseases, and to affect the proliferation of non-malignant myeloid cells. Preliminary data from the applicant`s laboratory revealed that in addition they increased the resistance of human myeloid cell lines to drugs used in the therapy of AML. In the course of the proposed work, the presumed role of CALCRL and its ligands in augmenting drug resistance of malignant myeloid cells shall be investigated in depth. Several complementary cell line models shall be established, and the ability of ADM and CGRP to confer protection from chemotherapy induced cell death in a CALCRL dependent manner shall be scrutinized. Key results will be confirmed in primary cells from patients with AML. Immunoblot analyses and RNA based next generation sequencing shall be used to investigate the downstream pathways mediating the biological effects of CALCRL activation. Finally, the role of CALCRL in mediating resistance to antileukemic therapy shall be confirmed in an appropriate mouse model. If these studies should corroborate the suspected role of CALCRL in mediating drug resistance in AML, this receptor may be considered as a novel target for rationally designed drugs that may be used in the treatment of relapsed AML.

Background: Acute myeloid leukemia (AML) is a malignant disease of blood cells that is often fatal. It is caused by tumor cell specific alterations of the genetic material ("mutations"), as well as changes in the activity ("expression") of certain genes. A specialized sub-population of AML cells, the leukemic stem cells, are responsible for disease emergence, therapy resistance, and relapse. Recently, some drugs have been approved that act specifically against mutations and gene expression changes that are causative for AML. Nevertheless, classical chemotherapy remains an important pillar of treatment for the majority of patients. In most patients, chemotherapy can dramatically reduce disease burden, thus leading to a remission. However, disease recurrence (relapse) is frequent, and its treatment is rarely successful. Identification of mutations and/or gene expression changes contributing to relapse, and understanding their mechanism of action, may lead to further improvements in AML therapy. Our previous work has shown that expression, rather than mutation, of specific genes changes consistently between diagnosis and relapse of AML. Among the genes that are up-regulated at relapse is CALCRL, which has been reported to play a tumor-promoting role in other cancers. CALCRL also plays a role in migraines, and drugs inhibiting its activity have already been developed. We therefore aimed to investigate the role of CALCRL in AML. In an extension of the original project plan, two other genes that are de-regulated at relapse, namely, SOCS2 (up-regulated) and MTSS1 (down-regulated), were also subjected to functional characterization in AML. Results: CALCRL and SOCS2 are up-regulated at relapse of AML, and patients who already exhibit higher expression of these genes at diagnosis experience shorter survival. Conversely, MTSS1 is down-regulated at relapse, and patients with lower expression at diagnosis tend to live shorter. These observations were made by analysing anonymized publicly available data sets, and further support the possible roles of these genes in disease aggressiveness and therapy resistance. Moreover, experiments with cell lines and with mouse models of AML showed that up-regulation of CALCRL and SOCS2, and down-regulation of MTSS1, promoted disease aggressiveness, chemotherapy resistance, and leukemic stem cell-related properties in AML. Conclusion: The approach to identify genes that contribute to aggressiveness and therapy resistance of AML based on alterations of their expression between diagnosis and relapse was developed in the project leader's laboratory. The functional data obtained in the course of this project validate this approach, which may lead to the identification of novel therapeutic targets and thus, improved treatment of AML.