Cancer cell elimination using CRISPR-Cas9 editing of fusion genes

Cancer development involves several genetic mutations that can deregulate multiple genes. Targeting single genes is often insufficient to eliminate cancer cells, as it is the case for fusion oncogenes (FOs). FOs are chimeric genes resulting from in-frame fusions of the coding sequence of two genes involved in a chromosomal rearrangement [1]. FOs are classified as involving transcription factors or tyrosine kinases, and silence of its transcripts has been shown to inhibit tumor cell growth both in vitro and in vivo [2]. Furthermore, genome-wide mutational studies have provided additional support for some FOs as genetic drivers for cancer initiation [3, 4]. Another feature of FOs is that they are characterized by patient-specific breakpoints that occur in intronic regions, rarely disrupting coding sequences. 

It has been described that FOs drive the development of more than 16% human cancers [5], including mesodermal ones such as leukemias, lymphomas and sarcomas and epithelial ones such as prostate, colorectal and breast cancers or melanoma [6-8]; and ~350 FOs involving more than 300 genes have been identified [9]. Although FOs are attractive targets for directed therapy, their targeting has remained challenging due to the difficulties in specifically recognizing and targeting the resultant chimeric protein, and also because their products are intracellular, necessitating effective intracellular approaches for the delivery of therapeutic molecules targeting the chimeric transcripts or proteins. In this regard, several approaches have been used for FO proteins targeting such as small molecules, intrabodies and aptamers, as well as for FO transcripts in the form of antisense RNA, ribozymes and interference RNA.

 Figure 1: Oncogenic fusion genes and their products. Neckles C, et al. Fusion transcripts: Unexploited vulnerabilities in cancer? Wiley Interdiscip Rev RNA. 2020 Jan;11(1):e1562. doi: 10.1002/wrna.1562.

In the present, the development of genome editing approaches offers new possibilities to directly target and modify the genomic sequence of cancer cells. Published in Nature Communications [10], the laboratory of Sandra Rodríguez (CNIO, Spain) has developed a new genome editing strategy for targeting FOs which efficiently directed the elimination of cancer cells harboring a given FO. This method is based on targeting two intronic sequences, one on each of the genes involved in the FO, that induces a cancer cell-specific deletion that eliminates key protein domains or changes the reading frame of the FO. The most remarkable advantage is that this method induces deletion only in the cells harboring a FO, without affecting exonic sequences or protein expression of the germline non-rearranged alleles. The same guide RNAs allow the targeting of different isoforms or every patient-specific breakpoint of a given FO, and is thus a universal approach for cancer-associated FOs.


Figure 2: EWSR1-FLI1 fusion oncogene and guide RNA design. Two pairs of gRNAs were designed for efficient and specific FO targeting: one pair complementary to the intronic regions of EWSR1 (sgE3 and E6), and one pair complementary to the intronic regions of FLI1 (sgF6 and F8). Martinez-Lage, M., et al., In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells. Nat Commun, 2020. 11(1): p. 5060. 

A key factor for this approach is the targeting strategy to specifically disrupt FOs in cancer cells. For a successful editing two criteria must be met: it must not affect the exonic sequences or the expression of wild type alleles involved in the rearrangement, and it need to be feasible irrespective of the FO isoform or the patient-specific breakpoint. To validate the approach, researchers used a cellular model of Ewing sarcoma characterized by a chromosomal translocation that fuses EWSR1 (an RNA-binding protein) to FLI1 (a transcription factor that belongs to the ETS family). They designed a strategy to induce EWSR-FLI1 (EF)-specific genomic deletion targeting two genomic introns (one on each rearranged gene) flanking the breakpoint introns. Targeted introns were selected to generate large deletions including key functional domains of the FO, so the deletion will occur only in cells harboring the FO with both on-target intronic regions in the same chromosome. As intron-directed guide RNAs guarantee the germline configuration of non-rearranged EWSR1 and FLI1 alleles, the expression of wild type alleles is preserved in healthy cells. They found a robust loss of EF mRNA and protein at day 3 post-transduction, with no difference in proliferation rate in long-term culture when the guide RNAs were introduced in healthy cells. Therefore, the therapeutic targeting of the EF FO seems highly specific and does not interfere with non-rearranged alleles in human healthy cells.

To further test this tool in vivo, researchers used three patient-derived xenografts (PDX) models of Ewing sarcoma. The models were stablished by subcutaneous implantation of Ewing sarcoma PDXs into immunodeficient mice allowed to develop for 12 days. Tumors were > 74% smaller when treated with the gene editing approach, validating this tool as an in vivo approach to avoid cancer growth.

They also tested the possible synergy between the genomic editing approach together with doxorubicin, a commonly used chemotherapeutic option for Ewing sarcoma. In vitro, they saw a greater reduction of cancer cell viability (50%) when compared to single treatments (39% with gene editing and 42% with doxorubicin); these results were replicated in vivo, with a greater reduction in tumor size (92.12%) when combining the treatments, compared with monotherapy (83.7% for gene editing and 70.4% for doxorubicin). These results indicate that combined therapy is more effective than monotherapy, both in vitro and in vivo.


Figure 3: Diagram showing the approach used for in vivo treatment and survival curves. PDXs were implanted into immunosuppressed mice and tumors were allowed to develop. Then tumors were injected with the editing vector and analyzed 40 days after tumor implantation. Survival curve shows that mice that received the treatment had an increase survival rate than the control ones. Adapted from Martinez-Lage, M., et al., In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells. Nat Commun, 2020. 11(1): p. 5060.

This work support FOs as an ideal therapeutic target for the development of new directed cancer treatments due to their cancer-driving roles, their restriction to cancer cells and the reliance of tumors on them. CRISPR/Cas9 gene editing of FOs has proven to be a simple and efficient method for the specific elimination of cancer cells, having no impact on healthy tissue. Furthermore, the combination of CRISPR-based FO deletion together with chemotherapy agents has an additive/synergistic effect on cell viability, tumor growth and overall survival, providing an attractive strategy to eliminate cancer cells with high specificity and lower risks for the patients. 

Although CRISPR/Cas9-mediated genome editing could be a potential platform for cancer treatment, we need to bear in mind that this technology still has a few drawbacks to overcome such as its in vivo target efficiency, the potential off-target effects and the limitation of in vivo delivery into the cells. Even though, this new application of the CRISPR/Cas9 genome editing technique opens the door to the development of new strategies for cancer treatment.

We will keep our eyes wide open for the next innovation in the field of gene editing.

References

1. Rabbitts, T.H., Chromosomal translocations in human cancer. Nature, 1994. 372(6502): p. 143-9.

2. Thomas, M., J. Greil, and O. Heidenreich, Targeting leukemic fusion proteins with small interfering RNAs: recent advances and therapeutic potentials. Acta Pharmacol Sin, 2006. 27(3): p. 273-81.

3. Andersson, A.K., et al., The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias. Nat Genet, 2015. 47(4): p. 330-7.

4. Yoshihara, K., et al., The landscape and therapeutic relevance of cancer-associated transcript fusions. Oncogene, 2015. 34(37): p. 4845-54.

5. Gao, Q., et al., Driver Fusions and Their Implications in the Development and Treatment of Human Cancers. Cell Rep, 2018. 23(1): p. 227-238 e3.

6. Ahmed, A.A. and M. Abedalthagafi, Cancer diagnostics: The journey from histomorphology to molecular profiling. Oncotarget, 2016. 7(36): p. 58696-58708.

7. Chou, A., et al., NTRK gene rearrangements are highly enriched in MLH1/PMS2 deficient, BRAF wild-type colorectal carcinomas-a study of 4569 cases. Mod Pathol, 2020. 33(5): p. 924-932.

8. Tomlins, S.A., et al., Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science, 2005. 310(5748): p. 644-8.

9. Mertens, F., et al., The emerging complexity of gene fusions in cancer. Nat Rev Cancer, 2015. 15(6): p. 371-81.

10. Martinez-Lage, M., et al., In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells. Nat Commun, 2020. 11(1): p. 5060.

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