Key Points Dnmt3a-null hematopoietic stem cells (HSCs) cannot sustain long-term hematopoiesis. Cooperating c-Kit mutations drive leukemic transformation of Dnmt3a-null HSCs.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm resulting from the malignant transformation of T-cell progenitors, and comprises approximately 15% and 25% of pediatric and adult ALL cases respectively. It is well-established that activating NOTCH1 mutations are the major genetic lesions driving T-ALL in most patients, but efforts to develop targeted therapies against this pathway have produced limited success in decreasing leukemic burden and come with significant clinical side effects. A finer detailed understanding of the genetic and molecular mechanisms underlying T-ALL is required identify patients at increased risk for treatment failure and the development of precision medicine strategies. Generation of genetic models that more accurately reflect the normal developmental history of T-ALL are necessary to identify new avenues for treatment. The DNA methyltransferase enzyme DNMT3A is also recurrently mutated in T-ALL patients, and we show here that inactivation of Dnmt3a combined with Notch1 gain-of-function leads to an aggressive T-ALL in mouse models. Moreover, conditional inactivation of Dnmt3a in mouse hematopoietic cells leads to an accumulation of immature progenitors in the thymus which are less apoptotic. These data demonstrate that Dnmt3a is required for normal T-cell development, and acts as a T-ALL tumor suppressor.

Although a disease-modifying therapy for classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease) exists, poor understanding of cellular pathophysiology has hampered the development of more effective and persistent therapies. Here, we investigated the nature and progression of neurological and underlying neuropathological changes in Cln2 R207X mice, which carry one of the most common pathogenic mutations in human patients but are yet to be fully characterized. Long-term electroencephalography recordings revealed progressive epileptiform abnormalities, including spontaneous seizures, providing a robust, quantifiable, and clinically relevant phenotype. These seizures were accompanied by the loss of multiple cortical neuron populations, including those stained for interneuron markers. Further histological analysis revealed early localized microglial activation months before neuron loss started in the thalamocortical system and spinal cord, which was accompanied by astrogliosis. This pathology was more pronounced and occurred in the cortex before the thalamus or spinal cord and differed markedly from the staging seen in mouse models of other forms of neuronal ceroid lipofuscinosis. Neonatal administration of adeno-associated virus serotype 9–mediated gene therapy ameliorated the seizure and gait phenotypes and prolonged the life span of Cln2 R207X mice, attenuating most pathological changes. Our findings highlight the importance of clinically relevant outcome measures for judging preclinical efficacy of therapeutic interventions for CLN2 disease.

CLN1 disease is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by delivering monthly infusions of recombinant human PPT1 (rhPPT1) in PPT1-deficient mice (Cln1 −/− ), and CLN1 R151X sheep to assess scale up for translation. In Cln1 −/− mice, intracerebroventricular rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1 treated-mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1 R151X sheep, intracerebroventricular infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural magnetic resonance imaging and neuropathology. These findings demonstrate the feasibility and therapeutic efficacy of intracerebroventricular rhPPT1 enzyme replacement therapy. This represents a key step towards clinical testing of ERT in children with CLN1 disease and highlights the importance of a cross-species approach to developing a successful treatment strategy.

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