CRISPR Library Screening Applications in Research on Acute Leukemia

CRISPR Library Screening Applications in Research on Acute Leukemia

Acute leukemia can be divided into two types, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), and is one of the most common blood tumors in adults. Its onset is due to the malignant proliferation of hematopoietic stem cells and the loss of their ability to differentiate normally, inhibiting the normal function of hematopoietic cells, which results in severe consequences such as anemia, bleeding, and immunosuppression. In recent years, with the development of CRISPR technology, especially the application of CRISPR library screening, a large number of targets for acute leukemia-related treatments have been discovered. Various new types of targeted drugs such as IDH1/2 inhibitors, FLT3 inhibitors, BCL-2 inhibitors, and CPX-351 have gradually entered clinical trials, leading to a major shift in acute leukemia treatment from chemotherapy to targeted therapy^[1-2]. So how is CRISPR library screening applied in the research of acute leukemia? Today Ubigene has compiled several high-score papers, let’s learn from these paper and find out the answers.

1.Finding Genes Necessary for AML Cell Growth

For CRISPR library screening, the most direct application is to screen for genes necessary for cell growth. However, to ensure the reliability of the screening results, it is best to use multiple different cell lines and combine in vivo models for synchronous screening. Lin et al. selected 200 growth-dependent genes in AML cell lines from the database, and then constructed the corresponding AML cell growth-dependent gene knockout libraries. By using these libraries for screening two different AML cell lines, MV4-11 and U937, as well as a tumor xenograft model PDX (patient-derived xenograft), the researchers found that the SLC5A3 and MARCH5 genes were significantly down-regulated in all three library screening models (Figure 1). Further downstream mechanism studies by constructing AML cell lines in which these two genes were individually knocked out revealed that SLC5A3 supports the proliferation of AML cells via myo-inositol transport, while the MARCH5 gene prevents AML cells from undergoing apoptosis^[3].

Figure 1 The MARCH5 and SLC5A3 genes were significantly down-regulated in CRISPR library screening^[3]

2.Promotion of Immune Escape in AML Cells by SUSD6 Gene

In addition to screen for genes necessary for cell growth, using CRISPR library screening to identify drug-resistant genes is also a common method. Immune checkpoint therapy is a method of promoting anti-tumor immune responses by blocking inhibitory signals for T-cell activation. However, certain tumors exhibit primary resistance or develop adaptive resistance to immune checkpoint therapy, rendering it ineffective for the treatment of these types of tumors. To investigate the mechanisms of tumor cell resistance to immune checkpoint therapy, a research team focused on human AML cell line THP-1 and a mouse AML cell line RN2, and used a whole-genome knockout library combined with flow cytometry to screen for fluorescently labeled cells expressing high levels of tumor antigens and found significant enrichment of the SUSD6 gene in both AML cell lines (Figure 2). This suggests that knockout of the SUSD6 gene promotes the expression of tumor antigens, enhancing the efficacy of immune checkpoint therapy. Subsequent experiments involving SUSD6 gene knockout in cells and in vivo experiments confirmed that SUSD6 inhibits MHC-I expression in AML cells and its immunogenicity to promote immune escape responses in cells^[4].

Figure 2 The SUSD6 gene was enriched in cell populations expressing high levels of tumor antigens in two cell lines^[4]

Acute lymphoblastic leukemia can be further divided into two types: acute T lymphoblastic leukemia (T-ALL) and acute B lymphoblastic leukemia (B-ALL), which account for approximately 30% of acute leukemia. Although the incidence rate of ALL is lower than AML, compared with AML, ALL is more difficult to treat and has a poor prognosis. Currently, there is no FDA-approved treatment for ALL.

1.Significant Upregulation of Valine-tRNA Synthetase in T-ALL

tRNA biosynthesis disturbances are common in tumor cells; however, it is unknown whether tRNA biosynthesis disturbances occur in T-ALL. To investigate this issue, Thandapani et al. used a knockout library of tRNA biosynthesis-related genes to screen two different T-ALL cell lines, CUTLL1 and Jurkat, and found that the gene VARS, responsible for valine-tRNA synthesis, was significantly downregulated in these two cell lines (Figure 3). Analysis of T-ALL samples revealed significant upregulation of VARS mRNA expression. These results suggest that valine-tRNA biosynthesis is disrupted in T-ALL. To further validate the role of valine-tRNA biosynthesis, the researchers found that the knockout of VARS gene knockouts in cell lines or restricting valine intake of animals can significantly inhibit T-ALL proliferation and improve T-ALL symptoms (Figure 4)^[5].

Figure 3 The VARS gene was significantly downregulated in CRISPR library screening^[5]

Figure 4 Restricting valine intake significantly improved T-ALL mouse symptoms^[5]

2.NUDT21 Gene Negatively Regulates CD19 Expression in B-ALL

Lack of CD19 expression in B-ALL cells leads to resistance to CAR-T cell therapy, but how CD19 expression is regulated remains unknown. To explore this issue, Witkowski et al. used a whole-genome knockout library to screen three different B-ALL cell lines, Reh, 697, and NALM6. After performing flow cytometry to select for cell populations expressing high and low levels of CD19 and performing NGS sequencing analysis, NUDT21 was found to be significantly enriched in cell populations expressing high levels of CD19 in the three cell lines. It indicates that NUDT21 negatively regulates CD19 expression (Figure 5). After confirming high expression of NUDT21 in B-ALL, the research team used the CRISPR/Cas9 system to construct NUDT21 gene knockout B-ALL cell lines and found that NUDT21 directly inhibits the stability and protein expression of CD19 mRNA^[6].

Figure 5 NUDT21 was significantly enriched as a CD19 inhibitor^[6]

The development of CRISPR library screening technology has been ongoing for 10 years, and with the help of CRISPR library screening, numerous novel targets have been discovered in different fields, leading to the publication of many high-impact articles. Based on the above cases, it is easy to see that in recent years, high-impact articles published using CRISPR library screening generally have the following features in common: 1. Using multiple different cell lines for synchronous screening of a specific disease, and identifying targets through comparative analysis; 2. After identifying targets, constructing gene knockout cell lines for validation and conducting downstream mechanism studies. In addition, using smaller sgRNA libraries, combining flow cytometry sorting, and animal experiments are more likely to gain favor with reviewers for your research.

In order to help researchers conduct better research and publish high-quality articles, we have compiled a list of various cell lines commonly used in acute leukemia (Table 1) for your convenience. If you have research needs in acute leukemia, be sure to bookmark this information!

Table 1 Commonly Used Cell Lines in Acute Leukemia

* Ubigene provides CRISPR library screening services for popular cell lines such as THP-1, RAW264.7, Jurkat, etc. After identifying targets, you can validate them using Ubigene's cell bank, containing 4500+ KO cell lines, in which 300+ selected genes enjoy a special price of only $1780. Contact us for more details!

References

[1] Lin T, Liu D, Guan Z, Zhao X, Li S, Wang X, Hou R, Zheng J, Cao J, Shi M. CRISPR screens in mechanism and target discovery for AML. Heliyon. 2024 Apr 9;10(8):e29382.

[2] Kayser S, Levis MJ. Updates on targeted therapies for acute myeloid leukaemia. Br J Haematol. 2022 Jan;196(2):316-328.

[3] Lin S, Larrue C, Scheidegger NK, Seong BKA, Dharia NV, Kuljanin M, Wechsler CS, Kugener G, Robichaud AL, Conway AS, Mashaka T, Mouche S, Adane B, Ryan JA, Mancias JD, Younger ST, Piccioni F, Lee LH, Wunderlich M, Letai A, Tamburini J, Stegmaier K. An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML. Cancer Discov. 2022 Feb;12(2):432-449.

[4] Chen X, Lu Q, Zhou H, Liu J, Nadorp B, Lasry A, Sun Z, Lai B, Rona G, Zhang J, Cammer M, Wang K, Al-Santli W, Ciantra Z, Guo Q, You J, Sengupta D, Boukhris A, Zhang H, Liu C, Cresswell P, Dahia PLM, Pagano M, Aifantis I, Wang J. A membrane-associated MHC-I inhibitory axis for cancer immune evasion. Cell. 2023 Aug 31;186(18):3903-3920.e21.

[5] Thandapani P, Kloetgen A, Witkowski MT, Glytsou C, Lee AK, Wang E, Wang J, LeBoeuf SE, Avrampou K, Papagiannakopoulos T, Tsirigos A, Aifantis I. Valine tRNA levels and availability regulate complex I assembly in leukaemia. Nature. 2022 Jan;601(7893):428-433.

[6] Witkowski MT, Lee S, Wang E, Lee AK, Talbot A, Ma C, Tsopoulidis N, Brumbaugh J, Zhao Y, Roberts KG, Hogg SJ, Nomikou S, Ghebrechristos YE, Thandapani P, Mullighan CG, Hochedlinger K, Chen W, Abdel-Wahab O, Eyquem J, Aifantis I. NUDT21 limits CD19 levels through alternative mRNA polyadenylation in B cell acute lymphoblastic leukemia. Nat Immunol. 2022 Oct;23(10):1424-1432.