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Hematopoietic stem and progenitor cells (HSPCs) originate from the hemogenic endothelium via the endothelial-to-hematopoietic transition, are self-renewing, and replenish all lineages of blood cells throughout life. BCAS2 (breast carcinoma amplified sequence 2) is a component of the spliceosome and is involved in multiple biological processes. However, its role in hematopoiesis remains unknown. We established a bcas2 knockout zebrafish model by using transcription activator–like effector nucleases. The bcas2−/− zebrafish showed severe impairment of HSPCs and their derivatives during definitive hematopoiesis. We also observed significant signs of HSPC apoptosis in the caudal hematopoietic tissue of bcas2−/− zebrafish, which may be rescued by suppression of p53. Furthermore, we show that the bcas2 deletion induces an abnormal alternative splicing of Mdm4 that predisposes cells to undergo p53-mediated apoptosis, which provides a mechanistic explanation of the deficiency observed in HSPCs. Our findings revealed a novel and vital role for BCAS2 during HSPC maintenance in zebrafish.

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT–driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context.

Background: Primary central nervous system lymphoma (PCNSL) is a rare and aggressive subtype of B-cell lymphoma with very poor survival. Genetic alterations often occur in the chronic active B-cell receptor signaling which mediate the response to BTK inhibition (BTKi) in PCNSL. However, the efficacy of high-dose methotrexate as a first-line treatment was suboptimal with a short effective remission time and low response rate. We aimed to investigate the efficacy of induction treatment, high-dose methotrexate in combination with ibrutinib and temozolomide (MIT), in newly diagnosed PCNSL. Methods: Thirty-three patients were planned to be enrolled in this open-label, non-randomized, multi-center phase II prospective clinical study (NCT04514393). Between October 2020 and April 2021, nine newly diagnosed PCNSL patients were enrolled. Eligible patients are ≥18 and ≤75 years of age with adequate organ function and require at least one measurable lesion. The MIT induction treatment included six cycles of high-dose methotrexate (HD-MTX, 3.5 g/m 2, every three weeks), ibrutinib (560 mg/d, after HD-MTX clearance), and temozolomide (150 mg/m 2 d1-d5, every three weeks). After the completion of the six cycles of MIT induction treatment, ASCT consolidation (only for patients < 65 yr) and daily ibrutinib (560 mg/d) were administered up to two years, or until disease progression, intolerable toxicity, or death. Treatment response was evaluated by brain MRI and FDG-PET and/or cerebrospinal fluid (CSF) examination every two cycles of the induction MIT treatment. The primary objective of this study was to evaluate the overall response rate (ORR) of MIT induction treatment for PCNSL defined as the proportion of subjects with complete response (CR) or partial response (PR). The safety and toxicity of MIT treatment were also investigated. No dose reduction or delayed treatment occurred. Baseline tissue samples and sequential CSF/plasma samples were collected and underwent targeted next-generation sequencing (NGS). Results: The median age of the nine enrolled patients was 50 yr (range: 29-68) and six of them were male. Six patients have completed the six cycles of MIT treatment and the rest three had just completed two cycles. Hematologic toxicity was mild, Grade 3-4 neutropenia occurred in one patient. Other side effects such as opportunistic infections (including Aspergillus) were not observed. Eight out of the nine (88.9%) patients achieved CR. While patient #2 progressed and dropped out after two cycles of MIT treatment (Figure A). The mutational profiles of baseline tissue and/or CSF samples revealed that PIM1 and MYD88 mutations were present in 88.9% of the entire cohort followed by HISTHIE and CD78B (6/9, 66.7%). CDKN2A copy number loss was detected in four patients. Four patients with sequential CSF or plasma sampling during MIT treatment were analyzed for dynamic disease monitoring. As shown in the figure A, the clearance of ctDNA in the CSF/plasma samples was observed as early as post one cycle in three CR patients (#1,3,4) and maintained until the latest follow-up. However, the ctDNA remained detectable in the CSF sample of the PD patient (#2), which baseline genetic status shows MYD88 (M232T) mutation alone without CD79B mutations, and with CCND3, CDK4 and ERBB3 gene amplification(Figure B). After a median follow-up of 7 months, the 1-year PFS is 88.9% and the OS is 100%. Other clinical results of this ongoing study (NCT04514393) will be updated. Conclusions: The induction treatment of MIT achieved excellent responses in newly diagnosed PCNSL patients with mild hematologic toxicity. The clearance of ctDNA in CSF/plasma samples was observed during dynamic disease monitoring which was in accord with imageology-based response evaluation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The sterile-20 kinase misshapen/Nck-interacting kinase (NIK)-related kinase 1 (MINK1) is involved in many important cellular processes such as growth, cytoskeletal rearrangement, and motility. Here, with MINK1-deficient (MINK1(-/-)) mice, we showed that MINK1 plays an important role in hemostasis and thrombosis via the regulation of platelet functions. In the tail-bleeding assay, MINK1(-/-) mice exhibited a longer bleeding time than wild-type (WT) mice (575.2 ± 59.7 seconds vs 419.6 ± 66.9 seconds). In a model of ferric chloride-induced mesenteric arteriolar thrombosis, vessel occlusion times were twice as long in MINK1(-/-) mice as in WT mice. In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on a collagen matrix under arterial shear conditions was significantly reduced in MINK1(-/-) platelets. Moreover, MINK1(-/-) platelets demonstrated impaired aggregation and secretion in response to low doses of thrombin and collagen. Furthermore, platelet spreading on fibrinogen was largely hampered in MINK1(-/-) platelets. The functional differences of MINK1(-/-) platelets could be attributed to impaired adenosine 5'-diphosphate secretion. Signaling events associated with MINK1 appeared to involve extracellular signal-regulated kinase, p38, and Akt. Hence, MINK1 may be an important signaling molecule that mediates mitogen-activated protein kinase signaling and participates in platelet activation and thrombus formation.