Intellectual disability (ID) is a highly heterogeneous disorder involving at least 600 genes, yet a genetic diagnosis remains elusive in ∼35%-40% of individuals with moderate to severe ID. Recent meta-analyses statistically analyzing de novo mutations in >7,000 individuals with neurodevelopmental disorders highlighted mutations in PPM1D as a possible cause of ID. PPM1D is a type 2C phosphatase that functions as a negative regulator of cellular stress-response pathways by mediating a feedback loop of p38-p53 signaling, thereby contributing to growth inhibition and suppression of stress-induced apoptosis. We identified 14 individuals with mild to severe ID and/or developmental delay and de novo truncating PPM1D mutations. Additionally, deep phenotyping revealed overlapping behavioral problems (ASD, ADHD, and anxiety disorders), hypotonia, broad-based gait, facial dysmorphisms, and periods of fever and vomiting. PPM1D is expressed during fetal brain development and in the adult brain. All mutations were located in the last or penultimate exon, suggesting escape from nonsense-mediated mRNA decay. Both PPM1D expression analysis and cDNA sequencing in EBV LCLs of individuals support the presence of a stable truncated transcript, consistent with this hypothesis. Exposure of cells derived from individuals with PPM1D truncating mutations to ionizing radiation resulted in normal p53 activation, suggesting that p53 signaling is unaffected. However, a cell-growth disadvantage was observed, suggesting a possible effect on the stress-response pathway. Thus, we show that de novo truncating PPM1D mutations in the last and penultimate exons cause syndromic ID, which provides additional insight into the role of cell-cycle checkpoint genes in neurodevelopmental disorders.

Knl1 (also known as CASC5, UniProt Q8NG31) is an evolutionarily conserved scaffolding protein that is required for proper kinetochore assembly, spindle assembly checkpoint (SAC) function and chromosome congression. A number of recent reports have confirmed the prominence of Knl1 in these processes and provided molecular details and structural features that dictate Knl1 functions in higher organisms. Knl1 recruits SAC components to the kinetochore and is the substrate of certain protein kinases and phosphatases, the interplay of which ensures the exquisite regulation of the aforementioned processes. In this Commentary, we discuss the overall domain organization of Knl1 and the roles of this protein as a versatile docking platform. We present emerging roles of the protein interaction motifs present in Knl1, including the RVSF, SILK, MELT and KI motifs, and their role in the recruitment and regulation of the SAC proteins Bub1, BubR1, Bub3 and Aurora B. Finally, we explore how the regions of low structural complexity that characterize Knl1 are implicated in the cooperative interactions that mediate binding partner recognition and scaffolding activity by Knl1.

Background: Mature T cell lymphomas are aggressive, treatment resistant cancers that are associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy T cells. Unlike B cell depletion, pan T cell aplasia is prohibitively toxic. The mutually exclusive expression of TCR β chains (TRBC) 1 or 2 allows targeting the malignant T cell population while preserving T cell function. Notably, a two amino acid inversion is the only exposed and antibody-accessible feature differentiating the two isoforms, making the development of therapeutics targeting TRBC1 and TRBC2 challenging. We have previously described a TRBC1 specific antibody which has been incorporated into a Chimeric Antigen Receptor (CAR) (Maciocia et al, Nat Med, 2017). Here we describe the derivation and characterization of a TRBC2 specific binder and CAR. In addition, we investigate use of these selective binders as Antibody Drug Conjugates (ADCs) for the treatment of HTLV-1 associated leukemia/lymphoma for which CAR T cell therapy may not be well suited. Methods: Anti TRBC2 antibodies were derived through crystallography and structural engineering of the previously described TRBC1 specific antibody. Briefly, TRBC2 binders were obtained by screening of small libraries of the TRBC1 binder with randomizations in key residues identified by crystallographic data and in silico design. TRBC2 binder candidates were first optimized for CAR function. Subsequently, TRBC2 specific CAR T cells were evaluated in vitro and in vivo for anti-tumor activity and selectivity. TRBC1/TRBC2 targeting antibodies, were further characterized as ADCs for biophysical properties, antibody internalization and cytotoxic function. Results: In vitro testing of TRBC2 CARs showed comparable efficacy to the previously described TRBC1 CAR. TRBC2 CAR T cells were effective at killing TRBC2 target cells while sparing TRBC1 positive cells. In vivo mouse models demonstrated that both TRBC1 and TRBC2 directed CARs could target their respective antigens with a high degree of specificity. TRBC1 and TRBC2 specific antibodies were used to generate ADC molecules as a proof of concept. Anti-TRBC1/TRBC2 antibodies were internalized and showed potential as ADCs. Interestingly, we demonstrate that an optimal affinity window facilitates improved antibody uptake and have further engineered both our TRBC1 and TRBC2 antibodies to take advantage of this particularity. Conclusions: Following on from structural and library-based approaches to generate CAR T cells capable of specifically targeting TRBC2, we have further characterized TRBC2 specific CAR T cells in vitro and in vivo. We have shown that highly specific antibodies, engineered as targeting moieties for TRBC1 and TRBC2 CAR T cells, show promising characteristics for utility potentially also as ADCs, offering another modality through which this targeting paradigm can be exploited for the treatment of peripheral T cell lymphomas. Citation Format: Mathieu Ferrari, Vania Baldan, Priyanka Ghongane, Alex Nicholson, Reyisa Bughda, Zulaikha Akbar, Patrycja Wawrzyniecka, Paul Maciocia, Shaun Cordoba, Simon Thomas, Shimobi Onuoha, Martin Pule. Targeting TRBC1 and 2 for the treatment of T cell lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2183.