HOTTIP-dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia.

HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of ß-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced ß-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.

NPM1 mutation reprograms leukemic transcription network via reshaping TAD topology.

C-terminal mutation of Nucleophosmin 1 (NPM1C+) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, molecular mechanisms underlying NPM1C+-driven leukemogenesis remain elusive. Here, we report that NPM1C+ activates signature HOX genes and reprograms cell cycle regulators by altering CTCF-driven topologically associated domains (TADs). Hematopoietic-specific NPM1C+ knock-in alters TAD topology leading to disrupted regulation of the cell cycle as well as aberrant chromatin accessibility and homeotic gene expression, which results in myeloid differentiation block. Restoration of NPM1 within the nucleus re-establishes differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators that switch the oncogenic MIZ1/MYC regulatory axis in favor of interacting with coactivator NPM1/p300, and prevents NPM1C+-driven leukemogenesis. In sum, our data reveal that NPM1C+ reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation.

A coordinated function of lncRNA HOTTIP and miRNA-196b underpinning leukemogenesis by targeting FAS signaling.

MicroRNAs (miRNAs) may modulate more than 60% of human coding genes and act as negative regulators, whereas long noncoding RNAs (lncRNAs) regulate gene expression on multiple levels by interacting with chromatin, functional proteins, and RNAs such as mRNAs and microRNAs. However, the crosstalk between HOTTIP lncRNA and miRNAs in leukemogenesis remains elusive. Using combined integrated analyses of global miRNA expression profiling and state-of-the-art genomic analyses of chromatin such as ChIRP-seq (HOTTIP binding in genomewide), ChIP-seq, and ATAC-seq, we found that some miRNA genes are directly controlled by HOTTIP. Specifically, the HOX cluster miRNAs (miR-196a, miR-196b, miR-10a, and miR-10b), located cis and trans, were most dramatically regulated and significantly decreased in HOTTIP-/- AML cells. HOTTIP bound to the miR-196b promoter and HOTTIP deletion reduced chromatin accessibility and enrichment of active histone modifications at HOX cluster-associated miRNAs in AML cells, whereas reactivation of HOTTIP restored miR gene expression and chromatin accessibility in the CTCF-boundary-attenuated AML cells. Inactivation of HOTTIP or miR-196b promotes apoptosis by altering the chromatin signature at the FAS promoter and increasing FAS expression. Transplantation of miR-196b knockdown MOLM13 cells in NSG mice increased overall survival of mice compared to wild-type cells transplanted into mice. Thus, HOTTIP remodels the chromatin architecture around miRNAs to promote their transcription and consequently represses tumor suppressors and promotes leukemogenesis.

Sex Differences in Tolerance to Delta-9-Tetrahydrocannabinol in Mice With Cisplatin-Evoked Chronic Neuropathic Pain.

Tolerance to the pain-relieving effects of cannabinoids limits the therapeutic potential of these drugs in patients with chronic pain. Recent preclinical research with rodents and clinical studies in humans has suggested important differences between males and females in the development of tolerance to cannabinoids. Our previous work found that male mice expressing a desensitization resistant form (S426A/S430A) of the type 1 cannabinoid receptor (CB1R) show delayed tolerance and increased sensitivity to the antinociceptive effects of delta-9-tetrahydrocannabinol (∆9-THC). Sex differences in tolerance have been reported in rodent models with females acquiring tolerance to ∆9-THC faster than males. However, it remains unknown whether the S426A/S430A mutation alters analgesic tolerance to ∆9-THC in mice with chemotherapy-evoked chronic neuropathic pain, and also whether this tolerance might be different between males and females. Male and female S426A/S430A mutant and wild-type littermates were made neuropathic using four once-weekly injections of 5 mg/kg cisplatin and subsequently assessed for tolerance to the anti-allodynic effects of 6 and/or 10 mg/kg ∆9-THC. Females acquired tolerance to the anti-allodynic effects of both 6 and 10 mg/kg ∆9-THC faster than males. In contrast, the S426A/S430A mutation did not alter tolerance to ∆9-THC in either male or female mice. The anti-allodynic effects of ∆9-THC were blocked following pretreatment with the CB1R antagonist, rimonabant, and partially blocked following pretreatment with the CB2R inverse agonist, SR144528. Our results show that disruption of the GRK/ß-arrestin-2 pathway of desensitization did not affect sensitivity and/or tolerance to ∆9-THC in a chronic pain model of neuropathy.