BCL2L11 Induction Mediates Sensitivity to Src and MEK1/2 Inhibition in Thyroid Cancer.

Patients with advanced thyroid cancer, including advanced papillary thyroid cancer and anaplastic thyroid cancer (ATC), have low survival rates because of the lack of efficient therapies available that can combat their aggressiveness. A total of 90% of thyroid cancers have identifiable driver mutations, which often are components of the MAPK pathway, including BRAF, RAS, and RET-fusions. In addition, Src is a non-receptor tyrosine kinase that is overexpressed and activated in thyroid cancer, which we and others have shown is a clinically relevant target. We have previously demonstrated that combined inhibition of Src with dasatinib and the MAPK pathway with trametinib synergistically inhibits growth and induces apoptosis in BRAF- and RAS-mutant thyroid cancer cells. Herein, we identified the pro-apoptotic protein BCL2L11 (BIM) as being a key mediator of sensitivity in response to combined dasatinib and trametinib treatment. Specifically, cells that are sensitive to combined dasatinib and trametinib treatment have inhibition of FAK/Src, MEK/ERK, and AKT, resulting in the dramatic upregulation of BIM, while cells that are resistant lack inhibition of AKT and have a dampened induction of BIM. Inhibition of AKT directly sensitizes resistant cells to combined dasatinib and trametinib but will not be clinically feasible. Importantly, targeting BCL-XL with the BH3-mimeitc ABT-263 is sufficient to overcome lack of BIM induction and sensitize resistant cells to combined dasatinib and trametinib treatment. This study provides evidence that combined Src and MEK1/2 inhibition is a promising therapeutic option for patients with advanced thyroid cancer and identifies BIM induction as a potential biomarker of response.

The molecular biology of tubulinopathies: Understanding the impact of variants on tubulin structure and microtubule regulation.

Heterozygous, missense mutations in both α- and ß-tubulin genes have been linked to an array of neurodevelopment disorders, commonly referred to as "tubulinopathies." To date, tubulinopathy mutations have been identified in three ß-tubulin isotypes and one α-tubulin isotype. These mutations occur throughout the different genetic domains and protein structures of these tubulin isotypes, and the field is working to address how this molecular-level diversity results in different cellular and tissue-level pathologies. Studies from many groups have focused on elucidating the consequences of individual mutations; however, the field lacks comprehensive models for the molecular etiology of different types of tubulinopathies, presenting a major gap in diagnosis and treatment. This review highlights recent advances in understanding tubulin structural dynamics, the roles microtubule-associated proteins (MAPs) play in microtubule regulation, and how these are inextricably linked. We emphasize the value of investigating interactions between tubulin structures, microtubules, and MAPs to understand and predict the impact of tubulinopathy mutations at the cell and tissue levels. Microtubule regulation is multifaceted and provides a complex set of controls for generating a functional cytoskeleton at the right place and right time during neurodevelopment. Understanding how tubulinopathy mutations disrupt distinct subsets of those controls, and how that ultimately disrupts neurodevelopment, will be important for establishing mechanistic themes among tubulinopathies that may lead to insights in other neurodevelopment disorders and normal neurodevelopment.

TUBA1A tubulinopathy mutants disrupt neuron morphogenesis and override XMAP215/Stu2 regulation of microtubule dynamics.

Heterozygous, missense mutations in α- or ß-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation's impact at the molecular and cellular levels scale with the severity of brain malformation. Here, we focus on two mutations at the valine 409 residue of TUBA1A, V409I, and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and a decrease in the number of neurite retraction events in primary rat neuronal cultures. These neuronal phenotypes are accompanied by increased microtubule acetylation and polymerization rates. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends in budding yeast and ablate tubulin binding to TOG (tumor overexpressed gene) domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations disrupt microtubule regulation typically conferred by XMAP215 proteins during neuronal morphogenesis and migration, and this impact on tubulin activity at the molecular level scales with the impact at the cellular and tissue levels.

Proteins are molecules made up of long chains of building blocks called amino acids. When a mutation changes one of these amino acids, it can lead to the protein malfunctioning, which can have many effects at the cell and tissue level. Given that human proteins are made up of 20 different amino acids, each building block in a protein could mutate to any of the other 19 amino acids, and each mutations could have different effects. Tubulins are proteins that form microtubules, thin tubes that help give cells their shape and allow them to migrate. These proteins are added or removed to microtubules depending on the cell's needs, meaning that microtubules can grow or shrink depending on the situation. Mutations in the tubulin proteins have been linked to malformations of varying severities involving the formation of ridges and folds on the surface of the brain, including lissencephaly, pachygyria or polymicrogyria. Hoff et al. wanted to establish links between tubulin mutations and the effects observed at both cell and tissue level in the brain. They focused on two mutations in the tubulin protein TUBA1A that affect the amino acid in position 409 in the protein, which is normally a valine. One of the mutations turns this valine into an amino acid called isoleucine. This mutation is associated with pachygyria, which leads to the brain developing few ridges that are broad and flat. The second mutation turns the valine into an alanine, and is linked to lissencephaly, a more severe condition in which the brain develops no ridges, appearing smooth. Hoff et al. found that both mutations interfere with the development of the brain by stopping neurons from migrating properly, which prevents them from forming the folds in the brain correctly. At the cellular level, the mutations lead to tubulins becoming harder to remove from microtubules, making microtubules more stable than usual. This results in longer microtubules that are harder for the cell to shorten or destroy as needed. Additionally, Hoff et al. showed that the mutant versions of TUBA1A have weaker interactions with a protein called XMAP215, which controls the addition of tubulin to microtubules. This causes the microtubules to grow uncontrollably. Hoff et al. also established that the magnitude of the effects of each mutation on microtubule growth scale with the severity of the disorder they cause. Specifically, cells in which TUBA1A is not mutated have microtubules that grow at a normal rate, and lead to typical brain development. Meanwhile, cells carrying the mutation that turns a valine into an alanine, which is linked to the more severe condition lissencephaly, have microtubules that grow very fast. Finally, cells in which the valine is mutated to an isoleucine ­ the mutation associated with the less severe malformation pachygyria ­ have microtubules that grow at an intermediate rate. These findings provide a link between mutations in tubulin proteins and larger effects on cell movement that lead to brain malformations. Additionally, they also link the severity of the malformation to the severity of the microtubule defect caused by each mutation. Further work could examine whether microtubule stabilization is also seen in other similar diseases, which, in the long term, could reveal ways to detect and treat these illnesses.

Kinetically Stabilizing Mutations in Beta Tubulins Create Isotype-Specific Brain Malformations.

Mutations in the family of genes encoding the tubulin subunits of microtubules are associated with a spectrum of human brain malformations known as tubulinopathies. How these mutations impact tubulin activity to give rise to distinct developmental consequences is poorly understood. Here we report two patients exhibiting brain malformations characteristic of tubulinopathies and heterozygous T178M missense mutations in different ß-tubulin genes, TUBB2A or TUBB3. RNAseq analysis indicates that both TUBB2A and TUBB3 are expressed in the brain during development, but only TUBB2A maintains high expression in neurons into adulthood. The T178 residue is highly conserved in ß-tubulins and located in the exchangeable GTP-binding pocket of ß-tubulin. To determine the impact of T178M on ß-tubulin function we created an analogous mutation in the ß-tubulin of budding yeast and show that the substitution acts dominantly to produce kinetically stabilized microtubules that assemble and disassemble slowly, with fewer transitions between these states. In vitro experiments with purified mutant tubulin demonstrate that T178M decreases the intrinsic assembly activity of ß-tubulin and forms microtubules that rarely transition to disassembly. We provide evidence that the T178M substitution disrupts GTPase-dependent conformational changes in tubulin, providing a mechanistic explanation for kinetic stabilization. Our findings demonstrate the importance of tubulin's GTPase activity during brain development, and indicate that tubulin isotypes play different, important roles during brain development.

Bridging the Gap: The Importance of TUBA1A α-Tubulin in Forming Midline Commissures.

Developing neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of α/ß-tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment. TUBA1A constitutes the majority of α-tubulin in the developing brain and mutations to TUBA1A in humans cause severe brain malformations accompanied by varying neurological defects, collectively termed tubulinopathies. Studies of TUBA1A function in mammalian cells have been limited by the presence of multiple genes encoding highly similar tubulin proteins, which leads to α-tubulin antibody promiscuity and makes genetic manipulation challenging. Here, we test mutant tubulin levels and assembly activity and analyze the impact of TUBA1A reduction on growth cone composition, neurite extension, and commissural axon architecture during brain development. We present a novel tagging method for studying and manipulating TUBA1A in cells without impairing tubulin function. Using this tool, we show that a TUBA1A loss-of-function mutation TUBA1A N102D (TUBA1A ND ), reduces TUBA1A protein levels and prevents incorporation of TUBA1A into microtubule polymers. Reduced Tuba1a α-tubulin in heterozygous Tuba1a ND/+ mice leads to grossly normal brain formation except a significant impact on axon extension and impaired formation of forebrain commissures. Neurons with reduced Tuba1a as a result of the Tuba1a ND mutation exhibit slower neuron outgrowth compared to controls. Neurons deficient in Tuba1a failed to localize microtubule associated protein-1b (Map1b) to the developing growth cone, likely impacting stabilization of microtubules. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration and cortex development but not commissure formation, and provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment.

Inhibition of the CCL2 receptor, CCR2, enhances tumor response to immune checkpoint therapy.

Immunotherapies targeting the PD-1/PD-L1 axis are now a mainstay in the clinical management of multiple cancer types, however, many tumors still fail to respond. CCL2 is highly expressed in various cancer types and has been shown to be associated with poor prognosis. Inhibition or blockade of the CCL2/CCR2 signaling axis has thus been an area of interest for cancer therapy. Here we show across multiple murine tumor and metastasis models that CCR2 antagonism in combination with anti-PD-1 therapy leads to sensitization and enhanced tumor response over anti-PD-1 monotherapy. We show that enhanced treatment response correlates with enhanced CD8+ T cell recruitment and activation and a concomitant decrease in CD4+ regulatory T cell. These results provide strong preclinical rationale for further clinical exploration of combining CCR2 antagonism with PD-1/PD-L1-directed immunotherapies across multiple tumor types especially given the availability of small molecule CCR2 inhibitors and antibodies.