The mechanisms controlling axon guidance are of fundamental importance in understanding brain development. Growing corticospinal and somatosensory axons cross the midline in the medulla to reach their targets and thus form the basis of contralateral motor control and sensory input. The motor and sensory projections appeared uncrossed in patients with horizontal gaze palsy with progressive scoliosis (HGPPS). In patients affected with HGPPS, we identified mutations in the ROBO3 gene, which shares homology with roundabout genes important in axon guidance in developing Drosophila , zebrafish, and mouse. Like its murine homolog Rig1/Robo3, but unlike other Robo proteins, ROBO3 is required for hindbrain axon midline crossing.

There is a significant decrease in normal optic nerve diameter along its length in normal subjects, reflecting reduction in connective tissue. High-resolution MRI is a valid and sensitive method of detecting subtle changes in retrobulbar optic nerve size and can be useful in the investigation of structural optic nerve lesions. Optic nerve diameters must be measured, however, at similar distances posterior to the globe to allow meaningful comparisons.

The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming. Here, we report that mitochondrial apoptosis resistance in T cell acute lymphoblastic leukemia (T-ALL) is mediated by inactivation of polycomb repressive complex 2 (PRC2). In T-ALL clinical specimens, loss-of-function mutations of PRC2 core components (EZH2, EED, or SUZ12) were associated with mitochondrial apoptosis resistance. In T-ALL cells, PRC2 depletion induced resistance to apoptosis induction by multiple chemotherapeutics with distinct mechanisms of action. PRC2 loss induced apoptosis resistance via transcriptional up-regulation of the LIM domain transcription factor CRIP2 and downstream up-regulation of the mitochondrial chaperone TRAP1. These findings demonstrate the importance of mitochondrial apoptotic priming as a prognostic factor in T-ALL and implicate mitochondrial chaperone function as a molecular determinant of chemotherapy response.

Graphical AbstractHighlights d A genetic screen revealed synthetic lethality of Wnt activation and asparaginase d Wnt signaling induces asparaginase sensitivity by inhibiting protein degradation d GSK3a inhibition phenocopies Wnt-induced sensitization to asparaginase d This combination has potent activity against asparaginaseresistant leukemias SUMMARY Resistance to asparaginase, an antileukemic enzyme that depletes asparagine, is a common clinical problem. Using a genome-wide CRISPR/Cas9 screen, we found a synthetic lethal interaction between Wnt pathway activation and asparaginase in acute leukemias resistant to this enzyme. Wnt pathway activation induced asparaginase sensitivity in distinct treatment-resistant subtypes of acute leukemia, but not in normal hematopoietic progenitors. Sensitization to asparaginase was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits glycogen synthase kinase 3 (GSK3)-dependent protein ubiquitination and proteasomal degradation, a catabolic source of asparagine. Inhibiting the alpha isoform of GSK3 phenocopied this effect, and pharmacologic GSK3a inhibition profoundly sensitized drug-resistant leukemias to asparaginase. Our findings provide a molecular rationale for activation of Wnt/STOP signaling to improve the therapeutic index of asparaginase.

Colorectal cancer is driven by mutations that activate canonical WNT/ β -catenin sig naling, but inhibiting WNT has signifi cant on-target toxicity, and there are no approved therapies targeting dominant oncogenic drivers. We recently found that activating a β -cateninindependent branch of WNT signaling that inhibits GSK3-dependent protein degradation induces asparaginase sensitivity in drug-resistant leukemias. To test predictions from our model, we turned to colorectal cancer because these cancers can have WNT-activating mutations that function either upstream (i.e., R-spondin fusions) or downstream ( APC or β -catenin mutations) of GSK3, thus allowing WNT/ β -catenin and WNT-induced asparaginase sensitivity to be unlinked genetically. We found that asparaginase had little effi cacy in APC or β -catenin-mutant colorectal cancer, but was profoundly toxic in the setting of R-spondin fusions. Pharmacologic GSK3 α inhibition was suffi cient for asparaginase sensitization in APC or β -catenin-mutant colorectal cancer, but not in normal intestinal progenitors. Our fi ndings demonstrate that WNT-induced therapeutic vulnerabilities can be exploited for colorectal cancer therapy.

Resistance to asparaginase, an antileukemic enzyme that depletes asparagine, is a common clinical problem. Using a genome-wide CRISPR/Cas9 screen, we found a synthetic lethal interaction between Wnt pathway activation and asparaginase in acute leukemias resistant to this enzyme. Wnt pathway activation induced asparaginase sensitivity in distinct treatment-resistant subtypes of acute leukemia, including T-lymphoblastic, hypodiploid B-lymphoblastic, and acute myeloid leukemias, but not in normal hematopoietic progenitors. Sensitization to asparaginase was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3dependent protein ubiquitination and degradation. Inhibiting the alpha isoform of GSK3 phenocopied this effect, and pharmacologic GSK3 inhibition profoundly sensitized drug-resistant leukemias to asparaginase. Our findings provide a molecular rationale for activation of Wnt/STOP signaling to improve the therapeutic index of asparaginase. SIGNIFICANCEThe intensification of asparaginase-based therapy has improved outcomes for several subtypes of acute leukemia, but the development of treatment resistance has a poor prognosis. We hypothesized, from the concept of synthetic lethality, that gain-of-fitness alterations in drugresistant cells had conferred a survival advantage that could be exploited therapeutically. We found a synthetic lethal interaction between activation of Wnt-dependent stabilization of proteins (Wnt/STOP) and asparaginase in acute leukemias resistant to this enzyme. Inhibition of the alpha isoform of GSK3 was sufficient to phenocopy this effect, and the combination of GSK3-selective inhibitors and asparaginase had marked therapeutic activity against leukemias resistant to monotherapy with either agent. These data indicate that drug-drug synthetic lethal interactions can improve the therapeutic index of cancer therapy.

Magnetic resonance imaging (MRI) now enables precise visualization of the mechanical state of the living human orbit, enabling inferences about the effects of mechanical factors on ocular kinematics. We used 3-dimensional magnetic search coil recordings and MRI to investigate the mechanical state of the orbit during vergence in humans. Horizontal convergence of 23° from a remote to a near target aligned on one eye was geometrically ideal, and was associated with lens thickening and extorsion of the rectus pulley array of the aligned eye with superior oblique muscle relaxation and inferior oblique muscle contraction. There was no rectus muscle cocontraction. Subjective fusion through a 1° vertical prism caused a clockwise (CW) torsion in both eyes, as well as variable vertical and horizontal vergences that seldom corresponded to prism amount or direction. MRI under these conditions did not show consistent torsion of the rectus pulley array, but a complex pattern of changes in rectus extraocular muscle (EOM) crossections, consistent with co-contraction. Binocular fusion during vergence is accomplished by complex, 3D eye rotations seldom achieving binocular retinal correspondence. Vergence eye movements are sometimes associated with changes in rectus EOM pulling directions, and may sometimes be associated with co-contraction. Thus, extraretinal information about eye position would appear necessary to interpret binocular correspondence, and to avoid diplopia.

Asparaginase, a bacterial enzyme that depletes the nonessential amino acid asparagine, is an integral component of acute leukemia therapy. However, asparaginase resistance is a common clinical problem whose biologic basis is poorly understood. We hypothesized, based on the concept of synthetic lethality, that gain-of-fitness alterations in the drug-resistant cells had conferred a survival advantage that could be exploited therapeutically. To identify molecular pathways that promote fitness of leukemic cells upon treatment with asparaginase, we performed a genome-wide CRISPR/Cas9 loss-of-function screen in the asparaginase-resistant T-ALL cell line CCRF-CEM. Cas9-expressing CCRF-CEM cells were transduced with a genome-wide guide RNA library (Shalem et al. Science343, 84-87, 2014), treated with either vehicle or asparaginase (10 U/L), and guide RNA representation was assessed. Our internal positive control, asparagine synthetase, was the gene most significantly depleted in asparaginase-treated cells (RRA significance score = 1.56 x 10-7), followed closely by two regulators of Wnt signaling, NKD2 and LGR6 (RRA score = 6 x 10-6and 2.19 x 10-5, respectively). To test how these genes regulate Wnt signaling in T-ALL, we transduced CCRF-CEM cells with shRNAs targeting NKD2 or LGR6, or with an shLuciferase control. Knockdown of NKD2 or LGR6 increased levels of active β-catenin, as well as the activity of a TopFLASH reporter of canonical Wnt/β-catenin transcriptional activity (P < 0.0001), indicating that NKD2 and LGR6 are negative regulators of Wnt signaling in these cells. We then validated the screen results using shRNA knockdown of NKD2 or LGR6, which profoundly sensitized these cells to asparaginase (P< 0.0001) and potentiated asparaginase-induced apoptosis (P < 0.0001). Inhibition of glycogen synthase kinase 3 (GSK3) is a key event in Wnt-induced signal transduction. Thus, we tested whether CHIR99021, an ATP-competitive inhibitor of both GSK3 isoforms (GSK3α and GSK3β), could phenocopy the effect of Wnt pathway activation. Pharmacologic inhibition of GSK3 induced significant sensitization to asparaginase across a panel of cell lines representing distinct subtypes of treatment-resistant acute leukemia, including T-ALL, AML and hypodiploid B-ALL (Fig. 1a, b). Importantly, GSK3 inhibition did not sensitize normal hematopoietic progenitors to asparaginase, suggesting a leukemia-specific effect. Wnt-induced sensitization to asparaginase was independent of β-catenin and mTOR activation, because genetic and pharmacologic manipulation of these Wnt targets had no effect on asparaginase response. Instead, it was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3-dependent protein ubiquitination and proteasomal degradation (Acebron et al. Mol Cell54, 663-674, 2014, Taelman et al. Cell143, 1136-1148, 2010). Indeed,Wnt-induced sensitization to asparaginase was completely blocked by the transduction of leukemia cells with FBXW7 (P < 0.0001), whose overexpression can reverse Wnt/STOP (Acebron et al. Mol Cell54, 663-674; 2014), or by expression of a hyperactive proteasomal subunit ΔN-PSMA4 (P < 0.0001), which globally increases protein degradation (Choi et al. Nat Commun7, 10963, 2016). Although GSK3α and GSK3βare redundant for many of their biologic functions, genetic or pharmacologic inhibition of GSK3α fully phenocopied Wnt-induced sensitization to asparaginase (P < 0.0001), whereas selective inhibition of GSK3β had no effect. We then leveraged the recently developed GSK3α-selective small molecule inhibitor BRD0705 (Wagner et al. Sci Transl Med10, 2018) to test the in vivo therapeutic potential of our findings. Immunodeficient NRG mice were injected with leukemic cells from a primary asparaginase-resistant T-ALL patient derived xenograft, and treatment was begun after confirmation of leukemic engraftment (n=16 mice per group). In vivo, this PDX proved completely resistant to asparaginase or BRD0705 monotherapy, whereas the combination was highly efficacious (median survival of 17 days in vehicle, vs. median not reached at 60 days in combo-treated mice; P < 0.0001; Fig. 2a, b). The combination was also well-tolerated, with no appreciable weight changes or increases in serum bilirubin levels. Our findings provide a molecular rationale for activating Wnt/STOP signaling to improve the therapeutic index of asparaginase. Disclosures No relevant conflicts of interest to declare.