The Ability of CBFβ-SMMHC to Increase RUNX1 Binding Affinity to Target DNA Correlates with Its Leukemogenic Potential

Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. We recently showed that RUNX1 is indispensable for Cbfb-MYH11-induced leukemogenesis in a mouse model. We found that RUNX1 interacted with CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, to directly regulate critical genes for leukemogenesis (Zhen et al., Blood, 2020). However, our current understanding of the interaction between CBFβ-SMMHC and RUNX1 does not provide adequate explanation on how the RUNX1-CBFβ-SMMHC complex forms and how the complex interacts with DNA for leukemogenesis as CBFβ-SMMHC without the RUNX1 high-affinity-binding-domain (CBFβ-SMMHC-ΔHABD) is also able to induce leukemia while CBFβ-SMMHC with mutations in the C-terminal multimerization domain (CBFβ-SMMHC-mDE) is not able to induce leukemia in mice. To address this question, we used RHD domain of RUNX1, CBFβ, CBFβ-SMMHC, CBFβ-SMMHC-ΔHABD and CBFβ-SMMHC-mDE proteins, which were purified from E. coli overexpressing these proteins, to explore how the HABD and DE domains affect the interactions between CBFβ-SMMHC, RHD and RUNX1-target DNA Bio-Layer Interferometry (BLI) and negative staining. As expected, deletion of the HABD domain significantly reduced CBFβ-SMMHC's binding affinity to RHD by BLI assay. Interestingly, differences in binding affinity between RHD and different versions of CBFβ-SMMHC did not correlate with their leukemogenic capability. On the other hand, the binding affinity between RHD and its target oligo was more significantly enhanced by CBFβ-SMMHC and CBFβ-SMMHC-ΔHABD that can induce leukemia than CBFβ-SMMHC-DE, which cannot. We also found that both CBFβ-SMMHC and CBFβ-SMMHC-ΔHABD, but not CBFβ-SMMHC-mDE, could form a filament structure by negative staining, suggesting the filament formation ability is important for leukemogenesis by CBFβ-SMMHC. In addition, RHD reduces filament formation by CBFβ-SMMHC, which was overcome when target oligo was added. In contrast, RHD could not inhibit filament formation by CBFβ-SMMHC-ΔHABD, suggesting that HABD interaction is required for RHD to disrupt filament formation by CBFβ-SMMHC. Overall, we found that leukemogenic capability of CBFβ-SMMHC correlates with its ability to enhance binding between RHD and its target DNA and to form multimerized filaments. The results also suggest that HABD and DE domains of CBFβ-SMMHC are required for the formation of the RUNX1-CBFβ-SMMHC complex with higher binding affinity to target DNA. Disclosures No relevant conflicts of interest to declare.

Lysinibacillus massiliensis Isolated from the Synovial Fluid: A Case Report

Lysinibacillus massiliensis is an aerobic, endospore-forming, gram-negative staining bacterium with peritrichous flagella belonging to the Bacillaceae family. A few cases of L. massiliensis isolated from the cerebrospinal fluid and tissue have been reported. In this study, we aimed to describe a case of L. massiliensis isolated from the synovial fluid. The synovial fluid from a 74-year-old female patient was inoculated into blood culture bottle. Gram-negative rods were observed in a gram-stained smear from a positive blood culture bottle. The bacterium was identified as Lysinibacillus sphaericus/Lysinibacillus fusiformis, with a probability of 89% using an automated bacterial identification system (VITEK2; Biomerieux, France). Subsequently, 16S rRNA gene sequencing was performed, and the sequence was analyzed using the Basic Local Alignment Search Tool. The sequence had 99.9% (1426/1427) identity with the strain L. massiliensis (GenBank ID: NR_043092.1). To our knowledge, this is the first reported case of L. massiliensis isolated from the synovial fluid. When an endospore-forming gram-negative staining bacterium can not be identified by phenotypic characterization, L. massiliensis should be considered, and different microbiological methods should be used for identification.

Utility of ICG fluorescence imaging with vessel clamp for ileocecal resection while preserving ileal conduit constructed after previous total cystectomy

Background Indocyanine green (ICG) is useful for evaluating the intestinal perfusion of anastomosis. Especially for patients with prior surgeries, ICG imaging enables surgeons in visualizing the anatomical field. Here, we reported the positive and negative staining techniques of ICG fluorescence with vessel clamp for determining the optimal resection area of vessels and mesentery. Case presentation An 80-year-old man, who had an ileal conduit constructed after a prior total cystectomy, was diagnosed with ascending colon cancer. Although the tumor-feeding vessel was primarily the ileocecal artery, there was no detailed information about the blood running through the ileal conduit. At first, the ascending colon and the marginal vessels were transected at distal side of the tumor. Next, both, the ileocecal artery and the marginal artery of oral side of the ileal anastomotic site were clamped. Finally, we injected ICG intravenously to assess the blood flow. As a result, the blood flow between the ileal anastomotic site and transected ascending colon was not identified (negative staining). Therefore, we cut the root of the ileocecal artery, and dissected the peripheral mesocolon including the ileal anastomotic site. After the ileo-ascending colon anastomosis, we injected ICG intravenously again. The blood flow to the ileal conduit was preserved (positive staining). Conclusion ICG fluorescence imaging with vessel clamp can clearly visualize the demarcation line between ischemic and non-ischemic intestinal tract. In colorectal surgeries, this technique is useful to assess the anastomotic perfusion and determine optimal dissection area of vessels and mesentery in secondary intestinal surgery.