Loss of function of Atrx leads to activation of alternative lengthening of telomeres in a primary mouse model of sarcoma

Abstract

The development of a telomere maintenance mechanism is essential for immortalization in human cancer. While most cancers elongate their telomeres by expression of telomerase, 10-15% of human cancers use a pathway known as alternative lengthening of telomeres (ALT). In this work, we developed a genetically engineered primary mouse model of sarcoma in CAST/EiJ mice which displays multiple molecular features of ALT activation after CRISPR/Cas9 introduction of oncogenic Kras ^G12D and loss of function mutations of Trp53 and Atrx. In this model, we demonstrate that the loss of Atrx contributes to the development of ALT in an autochthonous tumor, and this process occurs independently of telomerase function by variation of mTR alleles. Furthermore, we find that telomere shortening from the loss of telomerase leads to higher chromosomal instability while loss of Atrx and activation of ALT lead to an increase in telomeric instability, telomere sister chromatid exchange, c-circle production, and formation of ALT-associated promyelocytic leukemia bodies (APBs). The development of this primary mouse model of ALT could enable future investigations into therapeutic vulnerabilities of ALT activation and its mechanism of action.

Figure 1:. Genetically Engineered Mouse Model of Soft Tissue Sarcoma With Loss of Atrx.

(A) CAST/EiJ mice expressing Cas9 (Rosa26–1loxp-Cas9/+) and either wild type mTR (mTR^+/+), heterozygous mTR loss (mTR^+/−), or homozygous mTR loss (mTR^−/−) are injected in the gastrocnemius muscle with a two-plasmid system to induce expression of oncogenic Kras^G12D and biallelic mutation of Trp53 (CAST KP model), with additional mutation of Atrx (CAST KPA model), followed by electroporation of the gastrocnemius muscle. Tumor initiation occurs approximately 60 days after electroporation of the plasmids. (B) Schematic of plasmid activity. The yellow plasmid contains a sgRNA targeting Kras, which is repaired with a homology directed repair template that induces an oncogenic Kras^G12D mutation. The blue plasmid contains a sgRNA targeting Trp53, and the red plasmid contains sgRNAs targeting Trp53 and Atrx. (C) mTR is an RNA template for mTERT which is essential for the function of telomerase (D) ATRX as an epigenetic regulator of chromatin. ATRX and DAXX deposit Histone 3.3 at repetitive elements, including telomeres.

Figure 2:. Amplicon Sequencing Predicts Atrx Expression.

(A) Genomic DNA was extracted from fresh frozen tumors and next generation amplicon sequencing was performed for the site of Atrx targeted by CRISPR/Cas9 sgRNA (blue text). Examples of wild-type, non-frameshift (NFS), frameshift (FS), and large deletion (LD) mutation sequences are shown. (B) Graphical representation of the proportion of amplicons in tumors and organization of tumors into wild type CAST KP which did not receive the sgRNA for Atrx, CAST KPA* which retain wild type function of Atrx after CRISPR/Cas9 editing, and CAST KPA which have loss of function mutations of Atrx. (C) Immunofluorescent staining of Atrx (red) and DAPI (blue) of primary cells from CAST KP, CAST KPA*, and CAST KPA.

Figure 3:. C-circle Measurement in Primary Tumors.

(A) Rolling circle amplification of c-circles was performed using DNA extracted from fresh frozen tumors. Chemiluminescent detection of dot blot products with and without the addition of phi29 DNA polymerase from CAST KP (blue), CAST KPA* (purple), and CAST KPA (red) and human control cell lines (black). (B) Graphical representation of the average c-circle content of tumors using a one-way ANOVA with Tukey’s modification.

Figure 4:. Quantifying ALT-associated PML bodies in Primary Tumors.

(A) Representative images of immunoFISH including DAPI (blue), PML (magenta), and telomere (yellow). The colocalization of ultra-bright telomere foci with PML protein is classified as an ALT-associated PML body (APB). (B) Graphical representation of the proportion of APB positive nuclei in individual tumors, compared using a Welch’s t-test.

Figure 5:. Cytogenetic Analysis of Primary Cell Lines.

(A) Metaphase Telomere FISH of primary cell lines, DAPI (blue) and telomere (yellow). (B-D) Graphical representation of (B) undetectable telomeres, (C) fragile telomeres, and (D) chromosome fusions. CAST KP (blue) CAST KPA (red). Statistics performed using one-way ANOVA with Sidak’s multiple comparisons. (E) Q-FISH violin plot of individual telomere fluorescence values from the TFL-Telo program, compared using a one-way ANOVA with Sidak’s multiple comparisons.

Figure 6:. Chromosome Orientation FISH (CO-FISH).

(A) Schematic of CO-FISH. (B) Representative images of CO-FISH metaphase performed on primary cell lines. (C) Graphical representation of tSCE quantification in each cell line. Statistics performed using a one-way ANOVA with Sidak’s multiple comparisons.