Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms.

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type I or type II. While type II CALR-mutant proteins retain many of the Ca2+ binding sites present in the wild-type protein, type I CALR-mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain unexplored. Here, we show that the loss of Ca2+ binding residues in the type I mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1α/XBP1 pathway of the unfolded protein response. Genetic or pharmacologic inhibition of IRE1α/XBP1 signaling induces cell death in type I mutant but not type II mutant or wild-type CALR-expressing cells, and abrogates type I mutant CALR-driven MPN disease progression in vivo. SIGNIFICANCE: Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265.

Loss of MEN1 function impairs DNA repair capability of pancreatic neuroendocrine tumors.

Somatic MEN1 mutations occur in up to 50% of pancreatic neuroendocrine tumors (PanNETs). Clinical studies have shown that radiation therapy (IR) is effective in a subset of PanNETs, but it remains unclear why some patients respond better to IR than others. Herein, we study whether MEN1 loss of function increases radiosensitivity of PanNETs and determine its effect on DNA double-strand break (DSB) repair. After creating a MEN1 knockout PanNET cell line, we confirmed reduced DSB repair capacity in MEN1-deficient cells and linked these findings to a defect in homologous recombination, as well as reduced BRCA2 expression levels. Consistent with this model, we found that MEN1 mutant cells displayed increased sensitivity to the highly trapping poly (ADP-ribose) polymerase (PARP) 1 inhibitor talazoparib in vitro. Our results suggest that combining IR with PARP inhibition may be beneficial in patients with PanNETs and MEN1 loss of function.

TdT-dUTP DSB End Labeling (TUDEL), for Specific, Direct In Situ Labeling of DNA Double Strand Breaks.

The genome of a living cell is continuously damaged by various exogenous and endogenous factors yielding multiple types of DNA damage including base damage and damage to the sugar-phosphate backbone of DNA. Double Strand Breaks (DSBs) are the most severe form of DNA damage and if left unchecked, may precipitate genomic rearrangements, cell death or contribute to malignancy. In clinical contexts, radiation is often used to induce DSBs as a form of genotoxic therapy. Despite the importance of DSBs and their repair, as yet there is no facile assay to detect DSBs in situ or to quantify their location or proximity to other cellular constituents. Such an assay would help to disentangle DDR signaling pathways and identify new molecular players involved in DSB repair. These efforts, in turn, may facilitate drug screening and accelerate the discovery of novel, more effective genotoxic agents. We have developed such an assay, presented here, and term it TdT-dUTP DSB End Labeling (TUDEL).TUDEL makes use of Terminal Deoxynucleotidyl Transferase (TdT), a template-independent DNA polymerase. TdT is commonly used in TUNEL assays to yield a binary output of DNA damage. We have adapted this approach, using TdT and EdUTP to label individual DNA double strand breaks in irradiated cells and detecting the incorporated EdU with fluorescent probes via Click chemistry. This tool complements and is compatible with existing, indirect methods to track DSBs such as immunofluorescent detection of γH2AX. TUDEL is also sufficiently specific, sensitive, quantitative, and robust to replace the neutral Comet assay for routine measurement of DSB formation and repair. Here we present a protocol for TUDEL.

Loss of a 7q gene, CUX1, disrupts epigenetically driven DNA repair and drives therapy-related myeloid neoplasms.

Therapy-related myeloid neoplasms (t-MNs) are high-risk late effects with poorly understood pathogenesis in cancer survivors. It has been postulated that, in some cases, hematopoietic stem and progenitor cells (HSPCs) harboring mutations are selected for by cytotoxic exposures and transform. Here, we evaluate this model in the context of deficiency of CUX1, a transcription factor encoded on chromosome 7q and deleted in half of t-MN cases. We report that CUX1 has a critical early role in the DNA repair process in HSPCs. Mechanistically, CUX1 recruits the histone methyltransferase EHMT2 to DNA breaks to promote downstream H3K9 and H3K27 methylation, phosphorylated ATM retention, subsequent γH2AX focus formation and propagation, and, ultimately, 53BP1 recruitment. Despite significant unrepaired DNA damage sustained in CUX1-deficient murine HSPCs after cytotoxic exposures, they continue to proliferate and expand, mimicking clonal hematopoiesis in patients postchemotherapy. As a consequence, preexisting CUX1 deficiency predisposes mice to highly penetrant and rapidly fatal therapy-related erythroleukemias. These findings establish the importance of epigenetic regulation of HSPC DNA repair and position CUX1 as a gatekeeper in myeloid transformation.