Analysis of lysine recognition and specificity of the Bacillus subtilis L box riboswitch.

The ever-changing environment of a bacterial cell requires sophisticated mechanisms to adjust gene expression in response to changes in nutrient availability. L box riboswitch RNAs regulate gene expression in response to cellular lysine (lys) concentrations in the absence of additional regulatory factors. In Bacillus subtilis, binding of lysine (lys) to the L box RNA causes premature transcription termination in the leader region upstream of the lysC coding sequence. To date, little is known about the specific RNA-lys interactions required for transcription termination. In this study, we characterize features of the B. subtilis lysC leader RNA responsible for lys specificity, and structural elements of the lys molecule required for recognition. The wild-type lysC leader RNA can recognize and discriminate between lys and lys analogs. We identified leader RNA variants with mutations in the lys-binding pocket that exhibit changes in the specificity of ligand recognition. These data demonstrate that lysC leader RNA specificity is the result of recognition of ligand features through a series of distinct interactions between lys and nucleotides that comprise the lys-binding pocket, and provide insight into the molecular mechanisms employed by L box riboswitch RNAs to bind and recognize lys.

Biologic and clinical analysis of childhood gamma delta T-ALL identifies LMO2/STAG2 rearrangements as extremely high-risk.

Acute lymphoblastic leukemia expressing the gamma delta T cell receptor (yo T-ALL) is a poorly understood disease. We studied 200 children with yo T-ALL from 13 clinical study groups to understand the clinical and genetic features of this disease. We found age and genetic drivers were significantly associated with outcome. yo T-ALL diagnosed in children under three years of age was extremely high-risk and enriched for genetic alterations that result in both LMO2 activation and STAG2 inactivation. Mechanistically, using patient samples and isogenic cell lines, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping, resulting in deregulation of gene expression associated with T-cell differentiation. High throughput drug screening identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which can be targeted by Poly(ADP-ribose) polymerase (PARP) inhibition. These data provide a diagnostic framework for classification and risk stratification of pediatric yo T-ALL.

Proteasome inhibition targets the KMT2A transcriptional complex in acute lymphoblastic leukemia.

Rearrangments in Histone-lysine-N-methyltransferase 2A (KMT2Ar) are associated with pediatric, adult and therapy-induced acute leukemias. Infants with KMT2Ar acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival of 38%. Herein we evaluate 1116 FDA approved compounds in primary KMT2Ar infant ALL specimens and identify a sensitivity to proteasome inhibition. Upon exposure to this class of agents, cells demonstrate a depletion of histone H2B monoubiquitination (H2Bub1) and histone H3 lysine 79 dimethylation (H3K79me2) at KMT2A target genes in addition to a downregulation of the KMT2A gene expression signature, providing evidence that it targets the KMT2A transcriptional complex and alters the epigenome. A cohort of relapsed/refractory KMT2Ar patients treated with this approach on a compassionate basis had an overall response rate of 90%. In conclusion, we report on a high throughput drug screen in primary pediatric leukemia specimens whose results translate into clinically meaningful responses. This innovative treatment approach is now being evaluated in a multi-institutional upfront trial for infants with newly diagnosed ALL.