Characterization of SLC40A1 in Acute Myeloid Leukemia by CRISPRi Gene Editing
(2023) KIMM05 20231Department of Immunotechnology
- Abstract
- Acute myeloid leukemia (AML) is characterized by an accumulation of immature blast cells, which outgrow the bone marrow and suppress normal blood formation. The condition is associated with poor survival, mainly caused by relapsed and refractory disease. Hence, new therapeutics are needed. Recently, SLC40A1 was identified as potentially important for AML cells in a pooled CRISPR interference screen. Data from the screen also suggested that major histocompatibility (MHC) class I molecules were downregulated upon SLC40A1 knockdown. In accordance with the “missing-self” hypothesis, it was speculated that the lower expression of MHC class I molecules would lead to increased killing of the leukemia cells by natural killer (NK) cells. The aim of... (More)
- Acute myeloid leukemia (AML) is characterized by an accumulation of immature blast cells, which outgrow the bone marrow and suppress normal blood formation. The condition is associated with poor survival, mainly caused by relapsed and refractory disease. Hence, new therapeutics are needed. Recently, SLC40A1 was identified as potentially important for AML cells in a pooled CRISPR interference screen. Data from the screen also suggested that major histocompatibility (MHC) class I molecules were downregulated upon SLC40A1 knockdown. In accordance with the “missing-self” hypothesis, it was speculated that the lower expression of MHC class I molecules would lead to increased killing of the leukemia cells by natural killer (NK) cells. The aim of this study was to validate the immune regulatory role of SLC40A1. A gene set enrichment analysis was performed, showing negative enrichment of the gene set MHC Protein Complex in the knockdown cells, strengthening that MHC class I molecules are downregulated by knockdown of SLC40A1. Lentiviral vectors silencing SLC40A1 were generated and validated by sanger sequencing. The AML cell line THP-1, expressing endonuclease deficient dCas9 coupled to a Krüppel-associated box (dCas9-KRAB) was transduced with the vectors and knockdown was validated at the transcriptional level. Several attempts were made to confirm knockdown at the protein level but failed, probably due to unspecific antibodies. Moreover, SLC40A1 knockdown did not affect viability or result in a reduction in HLA-pan staining nor increased sensitivity to NK cell killing. Taken together, the data does not support that SLC40A1 play an immune regulatory role in AML. (Less)
- Popular Abstract
- Acute myeloid leukemia (AML) is an aggressive type of blood cancer known for its poor prognosis. Even though improvements have recently been made within the field, contemporary therapies highly resemble the ones that have been in place for half a century. This involves high doses of chemotherapy, which causes many toxic side effects to the patients. Although most patients initially achieve remission, recurrence of the disease is mercilessly common, and it often happens in combination with the development of drug resistance. Needless to say, new therapeutics need to be developed to not only treat, but to effectively cure all patients with AML.
An ideal way of targeting the disease, is to utilize the patient’s own immune cells to kill off... (More) - Acute myeloid leukemia (AML) is an aggressive type of blood cancer known for its poor prognosis. Even though improvements have recently been made within the field, contemporary therapies highly resemble the ones that have been in place for half a century. This involves high doses of chemotherapy, which causes many toxic side effects to the patients. Although most patients initially achieve remission, recurrence of the disease is mercilessly common, and it often happens in combination with the development of drug resistance. Needless to say, new therapeutics need to be developed to not only treat, but to effectively cure all patients with AML.
An ideal way of targeting the disease, is to utilize the patient’s own immune cells to kill off the cancer cells. Natural killer (NK) cells are a type of immune cell that target damaged and malfunctioning cells and can therefore be solicited for this purpose. To prevent NK cells from attacking healthy cells, all cells in the body express certain kinds of proteins on the surface, known as human leukocyte antigens (HLAs). In this context, HLAs can be seen as flags that communicate to the NK cells that they belong to the same organism and should therefore not be targeted. Unfortunately, HLAs are also expressed by AML cells, which allow them to evade the NK cells. Is it possible to minimize the expression of HLAs and hereby encourage NK cells to eliminate AML cells?
By utilizing clustered regularly interspaced short palindromic repeats (CRISPR) technology, commonly known as the “genetic scissors”, it is possible to silence genes of interest and investigate further effects of the silencing. Previously, such a CRISPR mediated screen had been performed, identifying the gene SLC40A1 as relevant for AML cells. It seemed that upon silencing of SLC40A1, HLAs were expressed to a lesser extent on the cells. To investigate whether this would indeed encourage NK cells to eliminate the cancer cells, CRISPR technology was used to create so called knockdown cells, which are cells that are genetically modified to silence the expression of SLC40A1. The knockdown cells were monitored over 10 days to see whether the silencing of SLC40A1 affected the cells’ ability to survive, which was not the case. HLAs were then measured and compared to control cells; however, no difference could be seen. Even so, the knockdown cells were still grown in the presence of NK cells, to investigate whether an increase in killing could be observed. Unfortunately, the response to NK cells was no different in the knockdown cells than in the control cells, indicating that this approach of targeting AML is not feasible by knockdown of SLC40A1. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9130992
- author
- Ohrt Jensen, Josephine LU
- supervisor
- organization
- course
- KIMM05 20231
- year
- 2023
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Acute Myeloid Leukemia, SLC40A1, Ferroportin, CRISPRi
- language
- English
- id
- 9130992
- date added to LUP
- 2023-06-28 11:54:52
- date last changed
- 2023-06-28 11:54:52
@misc{9130992,
abstract = {{Acute myeloid leukemia (AML) is characterized by an accumulation of immature blast cells, which outgrow the bone marrow and suppress normal blood formation. The condition is associated with poor survival, mainly caused by relapsed and refractory disease. Hence, new therapeutics are needed. Recently, SLC40A1 was identified as potentially important for AML cells in a pooled CRISPR interference screen. Data from the screen also suggested that major histocompatibility (MHC) class I molecules were downregulated upon SLC40A1 knockdown. In accordance with the “missing-self” hypothesis, it was speculated that the lower expression of MHC class I molecules would lead to increased killing of the leukemia cells by natural killer (NK) cells. The aim of this study was to validate the immune regulatory role of SLC40A1. A gene set enrichment analysis was performed, showing negative enrichment of the gene set MHC Protein Complex in the knockdown cells, strengthening that MHC class I molecules are downregulated by knockdown of SLC40A1. Lentiviral vectors silencing SLC40A1 were generated and validated by sanger sequencing. The AML cell line THP-1, expressing endonuclease deficient dCas9 coupled to a Krüppel-associated box (dCas9-KRAB) was transduced with the vectors and knockdown was validated at the transcriptional level. Several attempts were made to confirm knockdown at the protein level but failed, probably due to unspecific antibodies. Moreover, SLC40A1 knockdown did not affect viability or result in a reduction in HLA-pan staining nor increased sensitivity to NK cell killing. Taken together, the data does not support that SLC40A1 play an immune regulatory role in AML.}},
author = {{Ohrt Jensen, Josephine}},
language = {{eng}},
note = {{Student Paper}},
title = {{Characterization of SLC40A1 in Acute Myeloid Leukemia by CRISPRi Gene Editing}},
year = {{2023}},
}