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

Therapy-related myeloid neoplasms (t-MN) are high-risk, late effects in cancer survivors with poorly understood pathogenesis. 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, phospho-ATM retention, subsequent γH2AX foci 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 post-chemotherapy. 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.

Differential DNA Methylation of the IMMP2L Gene in Families with Maternally Inherited 7q31.1 Microdeletions is Associated with Intellectual Disability and Developmental Delay

Most copy number variations (CNVs) in the human genome display incomplete penetrance with unknown underlying mechanisms. One such mechanism may be epigenetic modification, particularly DNA methylation. The <i>IMMP2L</i> gene is located in a critical region for autism susceptibility on chromosome 7q (AUTS1). The level of DNA methylation was assessed by bisulfite sequencing of 87 CpG sites in the <i>IMMP2L</i> gene in 3 families with maternally inherited 7q31.1 microdeletions affecting the <i>IMMP2L</i> gene alone. Bisulfite sequencing revealed comparable levels of DNA methylation in the probands, healthy siblings without microdeletions, and their fathers. In contrast, a reduced DNA methylation index and increased <i>IMMP2L</i> expression were observed in lymphocytes from the healthy mothers compared with the probands. A number of genes were upregulated in the healthy mothers compared to controls and downregulated in probands compared to mothers. These genes were enriched in components of the ribosome and electron transport chain, as well as oxidative phosphorylation and various degenerative conditions. Differential expression in probands and mothers with <i>IMMP2L</i> deletions relative to controls may be due to compensatory processes in healthy mothers with <i>IMMP2L</i> deletions and disturbances of these processes in probands with intellectual disability. The results suggest a possible partial compensation for <i>IMMP2L</i> gene haploinsufficiency in healthy mothers with the 7q31.1 microdeletion by reducing the DNA methylation level. Differential DNA methylation of intragenic CpG sites may affect the phenotypic manifestation of CNVs and explain the incomplete penetrance of chromosomal microdeletions.

Transformation of Acute Myeloid Leukemia with Deletion of Chromosome 7q and Additional Abnormalities in Chromosome 8 in a Patient with Essential Thrombocythemia

This is a case report of a 60-year-old male patient with essential thrombocythemia (ET) that progressed to acute myeloid leukemia (AML) in approximately 9 years. His platelet count decreased approximately 8 years after ET treatment with hydroxyurea (HU) and aspirin. The dose of HU was reduced because of suspected myelosuppression due to HU; however, myelosuppression did not improve. Bone marrow examination revealed myelofibrosis; therefore, ruxolitinib was administered. Approximately 1 year later, his leukocyte and blast counts in the peripheral blood increased; thus, ET was judged to have progressed to AML-myelodysplasia-related change. Induction chemotherapy and consolidation therapy were initiated; however, the patient unfortunately failed to achieve complete remission. We then continued to administer salvage chemotherapy; however, his general condition worsened, and he died from cerebral hemorrhage. The karyotype at the onset of ET was 46,XY, which changed to 47,XY,del(7q),+8 at the time of AML diagnosis. In addition, genetic testing revealed FLT-3 ITD mutation. His histopathological analysis showed subarachnoid and intraparenchymal hemorrhages and tumor cell infiltration into the cerebrum, brainstem, and cerebellum. In this case, deletion of the long arm of chromosome 7, additional abnormalities in chromosome 8, and FLT3-ITD mutation were confirmed as risk factors for having developed secondary AML for approximately 9 years and death from cerebral hemorrhage 1 year later.

Kmt2c Limits the Self-Renewal Capacity of Multiply Divided HSCs By Promoting Sensitivity to Interleukin-1

Under normal homeostatic conditions, adult hematopoietic stem cells (HSCs) are usually quiescent. Hematopoietic stress, such as blood loss, infection, inflammation, or chemotherapy, can drive HSCs into cycle. When adult HSCs divide multiple times, they lose self-renewal capacity. Inflammatory cytokines, such as interleukin-1 (IL-1), can accelerate the loss of HSC self-renewal capacity by activating PU.1 and promoting myeloid commitment. This raises the question of whether intrinsic tumor suppressor genes can modulate sensitivity to inflammatory cytokines, and whether loss of these tumor suppressors can allow HSCs to evade commitment programs that would otherwise limit self-renewal capacity. The KMT2C tumor suppressor is located on chromosome 7q within a region that is frequently deleted in myelodsplastic syndrome (MDS) and therapy-related acute myeloid leukemia (AML). It encodes MLL3, a histone methyltransferase that activates enhancer elements and promotes transcription. Haploid KMT2C deletion has previously been shown to activate self-renewal programs and accelerate AML formation. This raised the question of whether KMT2C/MLL3 regulates normal HSC self-renewal and whether KMT2C deletion conveys a selective advantage to HSCs in contexts that would otherwise deplete the HSC pool. By protecting HSCs from exhaustion, KMT2C deletions may indirectly facilitate 7q-deficient MDS/AML. To understand whether and how Kmt2c regulates HSC self-renewal, we developed novel germline and conditional Kmt2c knockout mouse alleles. Mono- and bi-allelic Kmt2c deletions led to a modest increase in adult HSC numbers and a significant reduction in committed hematopoietic progenitors (HPCs). Kmt2c deletions markedly enhanced HSC self-renewal capacity, but HSC proliferation rates were not altered. To mimic conditions that lead to therapy-related AML, we deleted a single Kmt2c allele in a minority of HSCs. We then tested whether the Kmt2c-deleted HSC population expanded, relative to wild type HSCs, under native and stressed conditions. Under native conditions, the percentage of Kmt2c-heterozygous HSCs remained stable. However, after several cycles of chemotherapy, the Kmt2c mutant HSCs predominated within the marrow. In mechanistic studies, RNA-sequencing showed that Kmt2c-deficient HSCs expressed genes associated with innate immune signaling, including the receptor for interleukin-1 (IL1R), at lower levels than wild type HSCs. This suggested that Kmt2c mutations might sustain self-renewal capacity in multiply divided HSCs by dampening IL-1 driven myeloid commitment. In support of this hypothesis, Kmt2c-deficient HSCs retained multilineage potential when they were cultured with IL-1, and they failed to activate JNK and p38 upon exposure to IL-1. Altogether, our data suggest a mechanism to explain how KMT2C deletions, in the context of larger 7q deletions, may promote therapy related MDS/AML. When HSCs acquire a KMT2C deletion, they can then escape IL-1-mediated exhaustion when they are driven into cycle by chemotherapy or other stressors. In lieu of chemotherapy-induced stress, the same clones may remain relatively indolent. Disclosures No relevant conflicts of interest to declare.

Deletions of Chromosome 7q Affect Nuclear Organization and HLXB9Gene Expression in Hematological Disorders

The radial spatial positioning of individual gene loci within interphase nuclei has been associated with up- and downregulation of their expression. In cancer, the genome organization may become disturbed due to chromosomal abnormalities, such as translocations or deletions, resulting in the repositioning of genes and alteration of gene expression with oncogenic consequences. In this study, we analyzed the nuclear repositioning of HLXB9 (also called MNX1), mapping at 7q36.3, in patients with hematological disorders carrying interstitial deletions of 7q of various extents, with a distal breakpoint in 7q36. We observed that HLXB9 remains at the nuclear periphery, or is repositioned towards the nuclear interior, depending upon the compositional properties of the chromosomal regions involved in the rearrangement. For instance, a proximal breakpoint leading the guanine-cytosine (GC)-poor band 7q21 near 7q36 would bring HLXB9 to the nuclear periphery, whereas breakpoints that join the GC-rich band 7q22 to 7q36 would bring HLXB9 to the nuclear interior. This nuclear repositioning is associated with transcriptional changes, with HLXB9 in the nuclear interior becoming upregulated. Here we report an in cis rearrangement, involving one single chromosome altering gene behavior. Furthermore, we propose a mechanistic model for chromatin reorganization that affects gene expression via the influences of new chromatin neighborhoods.