The immunomodulatory role of CALRETICULIN in acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) is one of the most common and deadliest form of blood cancer in adults, accounting for over 80% of acute leukemic diagnoses. Despite the introduction of new targeted therapies, survival rate remains low in adults, and new therapeutic approaches are needed. In a previous CRISPR screen performed by the Järås group, CALRETICULIN was identified as a regulator of human leucocyte antigen (HLA) class I expression. Normal levels of HLA are fundamental to protect the cells from the cytotoxic action of macrophages and natural killer cells (NK cells). Calreticulin is a lectin-like chaperone protein involved in the quality control of antigen presentation, acting as a stabilizer for the peptide loading complex in the endoplasmic reticulum. In this study we knocked out the gene coding for calreticulin using CRISPR-Cas9 technology to analyze its immunomodulatory role in association with HLA class I expression. Calreticulin-deficient cells showed reduced expression of HLA class I on the cell surface. Moreover, our data suggest that HLA class I proteins are retained intracellularly, when analyzed 6 days after transduction, indicating that the peptide loading complex function is suboptimal. At 13 days after transduction, the intracellular retention of calreticulin was no longer detectable, probably due to an endo-lysosomal compartments degradation. We then studied if calreticulin-deficient cells were sensitized for killing by macrophages and NK cells. Consistent with our hypothesis, both the phagocytosis process by macrophages and cytolytic activity by NK cells were enhanced when calreticulin was knocked out, probably due to the depletion of human leucocyte antigen. In summary, our data suggest that calreticulin plays a fundamental role in the immune evasion process, ensuring the survival of AML cells. This discovery could be exploited to develop new strategies that inhibit calreticulin on AML cells and thereby sensitize them for killing by the innate immune system.

Popular Abstract

Disarming the leukemic cells against the immune system

Acute myeloid leukemia (AML) is the most common and deadliest form of blood cancer in adults, accounting for over 60% of leukemic diagnoses. It is characterized by the proliferation of immature blood cells that interfere with their normal function. The treatment is often capable of reducing the disease burden until complete remission, but relapse and treatment resistance is still a big concern. One valuable strategy to treat this type of cancer is to study how our immune system fights (and gets fooled) by the malignant cells. This could unveil new strategies to improve the responsiveness of the immune system against AML.

Leukemia starts in the bone marrow where an immature white blood cell, called myeloblast, undergoes mutations in its DNA. These mutations prevent the white blood cell from properly become a mature and functional, and at the same time it causes an abnormal proliferation in the blood. The immature white cell population progressively crowd up the patient’s blood, leaving less and less space to the functional mature cells, disrupting the overall blood function.

For these reasons, patients experience a wide set of symptoms including anaemia (lack of healthy red blood cells), thrombocytopenia (low platelets count), increased risk of infections and internal bleeding. The 5-year survival rate in adults is about 30%, often due to an increased presence of DNA mutations and to a decreased tolerance to the conventional therapies. Chemotherapy is the gold standard treatment for AML showing high efficacy in achieving complete remission. Unfortunately, relapse rates are very high and despite the introduction of new targeted therapies, survival rate remains low.

Our body has the innate capacity to get rid of most of the threats to our health, relying on “soldiers” of the immune system that defend us from external and internal enemies. Some of these soldiers are macrophages, big cells capable of eating pathogens and defective/dying cells, and natural killer cells, that kills virus-infected and mutated cells. Both are inhibited by the presence on the target cell surface of proteins of a family called Major Histocompatibility Complex (MHC), that generally signals its health status. These proteins get onto the cell surface thank to a molecular machinery called Peptide Loading Complex (PLC), which stability is controlled, among others, by a protein called calreticulin.

The aim of the project is using the recent technology of CRIPSR-Cas9 to deactivate the gene of calreticulin. CRISPR-Cas9 is a molecular tool that is capable of cutting a specific (and selectable) portion of DNA (aided by a proper guide) and to introduce mutations that can disrupt a gene. Deactivation of calreticulin gene in a leukemic cell line was performed to study the effect on MHC presentation and the macrophage/natural killer cells activity. We showed that calreticulin-deficient AML cells are more vulnerable to these immune cells, probably thanks to the depletion of MHC on the cell surface. This discovery could potentially lead to new therapies that relies on unarming the leukemic cells of their defence mechanisms against the immune system.

Master’s Degree Project in Molecular Biology 60 credits 2022
Department of Biology, Lund University
Advisor: Marcus Järås
Department Clinical Genetics, BMC

• author: Ferro, Michele
• supervisor: Marcus Järås; Maria Neves
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20212
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9103267
• date added to LUP: 2022-11-16 15:46:38
• date last changed: 2022-11-16 15:46:38