Defining mechanisms that regulate the alternative lengthening of telomeres

Telomeres are repetitive DNA sequences found at the ends of eukaryotic chromosomes that help maintain genome stability. Telomeres shorten every time a cell divides, eventually inducing replicative senescence. To gain replicative immortality cancer cells establish mechanisms to maintain telomere length over many cell divisions. Around 10% of cancers do this using a recombination-based pathway called the Alternative Lengthening of Telomeres (ALT). ALT resembles a specific type of homology-directed repair called break-induced replication (BIR). Through this body of work, we aimed to better understand both the genetics underlying ALT positive cancers and the mechanistic basis of ALT. ALT positive cancers frequently carry loss of function mutations in the genes for ATRX/DAXX, which function to regulate heterochromatin. Recently, we identified a novel chromosomal fusion event in ALT positive osteosarcoma causing defects in DAXX function. Additionally, we identified several osteosarcoma tumors with wild-type ATRX/DAXX that had abnormalities in SLX4IP or SMARCAL1, proteins recently shown to regulate the ALT pathway. These data suggest that a more thorough understanding of the ALT mechanism may reveal additional factors that are defective in ALT positive tumors. Building on this, we aimed to further define the mechanism of ALT by investigating the DNA translocase RAD54 in the ALT pathway. During BIR, a broken DNA strand invades a homologous template, forming a structure called a displacement loop (D-loop) where a strand of template DNA is displaced to allow base pairing between the broken DNA strand and the homologous template. The D-loop recruits DNA polymerases, leading to extension and repair of the broken DNA strand. RAD54 is known to regulate both the formation and resolution of D-loops. In this work, we found that RAD54 promotes elongation at ALT telomeres by mediating branch migration and dissolution of the D-loop. D-loops formed at ALT telomeres must be resolved before mitosis to prevent the formation of ultra-fine anaphase bridges. These data demonstrate that by mediating D-loop migration RAD54 plays an important role in both promoting telomere elongation and maintaining genome stability in ALT cells. Together this body of work represents advances in defining both the genetic and mechanistic basis of ALT.2021-01-30T00:00:00

Methylation-associated partial down-regulation of mesothelin causes resistance to anti-mesothelin immunotoxins in a pancreatic cancer cell line.

Anti-mesothelin Pseudomonas exotoxin A-based recombinant immunotoxins (RITs) present a potential treatment modality for pancreatic ductal adenocarcinoma (PDAC). To study mechanisms of resistance, the sensitive PDAC cell line KLM-1 was intermittently exposed to the anti-mesothelin SS1-LR-GGS RIT. Surviving cells were resistant to various anti-mesothelin RITs (IC50s >1 μg/ml), including the novel de-immunized RG7787. These resistant KLM-1-R cells were equally sensitive to the anti-CD71 HB21(Fv)-PE40 RIT as KLM-1, indicating resistance was specific to anti-mesothelin RITs. Mesothelin gene expression was partially down-regulated in KLM-1-R, resulting in 5-fold lower surface protein levels and decreased cellular uptake of RG7787 compared to KLM-1. Bisulfite sequencing analysis found that the mesothelin promoter region was significantly more methylated in KLM-1-R (59 ± 3.6%) compared to KLM-1 (41 ± 4.8%), indicating hypermethylation as a mechanism of mesothelin downregulation. The DNA methyltransferase inhibitor 5-azacytidine restored original mesothelin surface expression to more than half in KLM-1-R and increased sensitivity to RG7787 (IC50 = 722.4 ± 232.6 ng/ml), although cells remained significantly less sensitive compared to parental KLM-1 cells (IC50 = 4.41 ± 0.38 ng/ml). Mesothelin cDNA introduction in KLM-1-R led to 5-fold higher surface protein levels and significantly higher RG7887 uptake compared to KLM-1. As a result, the original sensitivity to RG7787 was fully restored (IC50 = 4.49 ± 1.11 ng/ml). A significantly higher RG7787 uptake was thus required to reach the original cytotoxicity in resistant cells, hinting that intracellular RIT trafficking is also a limiting factor. RNA deep sequencing analysis of KLM-1 and KLM-1-R cells supported our experimental findings; compared to KLM-1, resistant cells displayed differential expression of genes linked to intracellular transport and an expression pattern that matched a more general hypermethylation status. In conclusion, resistance to anti-mesothelin RITs in KLM-1 is linked to a methylation-associated down-regulation of mesothelin, while aberrations in RIT trafficking could also play a role

Activity of anti-mesothelin, anti-CD25 and anti-CD71 immunotoxins in KLM-1 and KLM-1-R.

<p><b>A</b>: KLM-1 and resistant KLM-1 (KLM-1-R) cells were incubated for 72 hrs with the anti-mesothelin SS1-LR-GGS, RG7787 or anti-CD25 LMB-2 as a control. Growth inhibition was evaluated with an ATP cell viability assay. With IC₅₀s below 10 ng/ml, KLM-1 is sensitive to the anti-mesothelin RITs, which is not the case for KLM-1-R (IC₅₀s > 1 μg/ml). 1 μg/ml LMB-2 decreased cell viability, indicating that this RIT concentration induces non-specific uptake. <b>B</b>: KLM-1 and KLM-1-R cells were incubated for 72 hrs with 100 or 1000 ng/ml RG7787. Apoptosis was evaluated with the Annexin V-PE Apoptosis Detection Kit I. RG7787 induces a significant increase in apoptotic KLM-1 cells, whereas KLM-1-R cells shows no meaningful increase in apoptosis. <b>C</b>: KLM-1 and KLM-1-R cells were incubated for 72 hrs with HB21(Fv)-PE40. Growth inhibition was evaluated with an ATP cell viability assay. Both cell lines are highly sensitive to this RIT.</p

Mesothelin downregulation is associated with CpG hypermethylation and mesothelin transfection restores sensitivity to of KLM-1-R to RG7787.

<p><b>A</b>: AZA leads to a 2.8-fold increase of mesothelin surface expression in resistant KLM-1 (KLM-1-R). Flow cytometry histogram of KLM-1, KLM-1-R, and KLM-1-R-AZA. Filled histograms represent secondary antibody controls. <b>B</b>: Three weeks of incubation with AZA, a DNA methyltransferase inhibitor, increases sensitivity to RG7787. KLM-1, KLM-1-R, and the AZA-treated cells (KLM-1-AZA and KLM-1-R-AZA) were treated for 72 hrs with RG7787. Growth inhibition was evaluated with an ATP cell viability assay. Dotted lines represent AZA-treated cells. <b>C</b>: CpGs in a region upstream of the mesothelin transcription start site are more methylated in KLM-1-R than in KLM-1. Three weeks of incubation with AZA decreases methylation in KLM-1-R cells. The analyzed region is located at chr16:808890-808742 and spans 147 bp and seven CpGs. <b>D</b>: Mesothelin transfection in KLM-1-R results in significant overexpression of mesothelin compared to KLM-1 (5-fold) and KLM-1-R (23-fold). Flow cytometry surface mesothelin levels in KLM-1, KLM-1-R and mesothelin-transfected resistant cells (KLM-1-R-<i>Msln</i>). <b>E</b>: Mesothelin overexpression in KLM-1-R restores sensitivity to RG7787. KLM-1 and KLM-1-R-<i>Msln</i> cells were incubated for 72 hrs with RG7787. Growth inhibition was evaluated with an ATP cell viability assay.</p

Mesothelin expression and RG7787 uptake is decreased in KLM-1-R.

<p><b>A</b>: Surface mesothelin levels are 5-fold lower in resistant KLM-1 (KLM-1-R) compared to KLM-1. Mesothelin expression of KLM-1, KLM-1-R and mesothelin-negative A431 cells (negative control) were evaluated using flow cytometry. Filled histograms are secondary antibody controls. <b>B</b>: Whole mesothelin protein level is decreased in KLM-1-R. Precursor (72 kDa) and cleaved mature mesothelin (37 kDa) were present in KLM-1. In KLM-1-R, the cleaved portion was detected at low levels. Protein levels were probed in untreated KLM-1 and KLM-1-R cell lysate by Western blot. β-actin acts as loading control. <b>C</b>: At each time point, cellular uptake of RG7787-Alexa647 in KLM-1 is significantly higher than in KLM-1-R, and significantly lower than in KLM-1-R-<i>Msln</i> (transfected with mesothelin). Uptake was evaluated at 30, 75 and 150 min. Average geomean fluorescence intensities converted into amount of RG7787 molecules.</p

Significance level for differentially expressed gene sets from KLM-1 to KLM-1-R.

<p>Table was generated by STRING using GO, KEGG, and Reactome databases, as indicated under dataset reference.</p><p>§Number of genes in our data set that are also present in the respective data-set list or pathway list.</p><p>*p-value represents the probability that 478 genes would show the distribution to match the same number of gene hits in the respective list. Datasets are ranked according to p-value. P < 0.001 is considered statistically significant.</p><p>Significance level for differentially expressed gene sets from KLM-1 to KLM-1-R.</p

Supplementary Figures from Synergistic Drug Combinations Promote the Development of Resistance in Acute Myeloid Leukemia

Supplementary Figures 1-5</p

Supplementary Table 1 from Synergistic Drug Combinations Promote the Development of Resistance in Acute Myeloid Leukemia

Supplementary Table 1 - Materials</p

SLX4IP Antagonizes Promiscuous BLM Activity during ALT Maintenance

International audienc