TGFß pathway is required for viable gestation of Fanconi anemia embryos.

Overexpression of the TGFß pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFß promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFß signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFß pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFß and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFß-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFß-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFß-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis.

Targeting DNA Repair with Combined Inhibition of NHEJ and MMEJ Induces Synthetic Lethality in TP53-Mutant Cancers.

DNA repair pathway inhibitors are a new class of anticancer drugs that are advancing in clinical trials. Peposertib is an inhibitor of DNA-dependent protein kinase (DNA-PK), which is a key driver of nonhomologous end-joining (NHEJ). To identify regulators of response to peposertib, we performed a genome-wide CRISPR knockout screen and found that loss of POLQ (polymerase theta, POLθ) and other genes in the microhomology-mediated end-joining (MMEJ) pathway are key predictors of sensitivity to DNA-PK inhibition. Simultaneous disruption of two DNA repair pathways via combined treatment with peposertib plus a POLθ inhibitor novobiocin exhibited synergistic synthetic lethality resulting from accumulation of toxic levels of DNA double-strand break end resection. TP53-mutant tumor cells were resistant to peposertib but maintained elevated expression of POLQ and increased sensitivity to novobiocin. Consequently, the combination of peposertib plus novobiocin resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models. Thus, the combination of a targeted DNA-PK/NHEJ inhibitor with a targeted POLθ/MMEJ inhibitor may provide a rational treatment strategy for TP53-mutant solid tumors. SIGNIFICANCE: Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia.

Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-ß pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.

Experience Informs Consummatory Choices for Congruent and Incongruent Odor-Taste Mixtures in Rats.

Experience is an essential factor informing food choice. Eating food generates enduring odor-taste associations that link an odor with a taste's quality and hedonic value (pleasantness/unpleasantness) and creates the perception of a congruent odor-taste combination. Previous human psychophysical experiments demonstrate that experience with odor-taste mixtures shapes perceptual judgments related to the intensity, familiarity, and pleasantness of chemosensory stimuli. However, how these perceptual judgments inform consummatory choice is less clear. Using rats as a model system and a 2-bottle brief-access task, we investigated how experience with palatable and unpalatable odor-taste mixtures influences consummatory choice related to odor-taste congruence and stimulus familiarity. We found that the association between an odor and a taste, not the odor's identity or its congruence with a taste, informs consummatory choice for odor-taste mixtures. Furthermore, we showed that the association between an odor and a taste, not odor neophobia, informs consummatory choice for odors dissolved in water. Our results provide further evidence that the association between an odor and a taste, after odor-taste mixture experience, is a fundamental feature guiding consummatory choice.

Experience-Dependent c-Fos Expression in the Mediodorsal Thalamus Varies With Chemosensory Modality.

The mediodorsal thalamus is a higher order thalamic nucleus critical for many cognitive behaviors. Defined by its reciprocal connections with the prefrontal cortex, the mediodorsal thalamus receives strong projections from chemosensory cortical areas for taste and smell, gustatory cortex and piriform cortex. Recent studies indicate the mediodorsal thalamus is involved in experience-dependent chemosensory processes, including olfactory attention and discrimination and the hedonic perception of odor-taste mixtures. How novel and familiar chemosensory stimuli are represented within this structure remains unclear. Here, we compared the expression of c-Fos in the mediodorsal thalami of rats familiar with an odor, a taste, or an odor-taste mixture with those that sampled the stimuli for the first time. We found that familiar tastes or odor-taste mixtures induced significantly greater c-Fos expression in the mediodorsal thalamus than novel tastes or odor-taste mixtures, whereas novel odors induced greater c-Fos expression than familiar odors. These experience-dependent and modality-specific differences in c-Fos expression may relate to the behavioral relevance of the chemosensory stimulus, including odor neophobia. In a two-bottle brief-access preference task, rats preferred water to isoamyl acetate-odorized water over multiple days. However, after experience with isoamyl acetate mixed with sucrose (odor-taste mixture), the preference for water was eliminated. These findings demonstrate that experience with chemosensory stimuli modulates responses in the mediodorsal thalamus, suggesting this structure plays an integral role in communicating behaviorally relevant chemosensory information to higher order areas to guide food-related behaviors.