B cell receptor (BCR) signaling and T cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can utilize microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short non-coding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs. CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22), but also other candidates for a role in microenvironmental interactions. Notably, all three miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation, and significantly shorter overall survival of CLL patients. We identified Tumor-Necrosis Factor Receptor-Associated Factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream NFkB signaling. In CLL, BCR-represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40-NFkB signaling. This regulatory loop is disrupted by "BCR inhibitors" (BTK inhibitor ibrutinib or PI3K inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29-TRAF4-CD40 signaling axis modulated by the BCR activity.
Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for BCR inhibitors such as ibrutinib or idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how precisely CLL cells regulate their homingintegrate multiple migratory signals while balancing survival in peripheral blood and the decision to return to immune niches. Here we provide evidence for the use of CXCR4/CD5 intraclonal subpopulations to study CLL cells migration regulation. We performed RNA profiling of CXCR4dimCD5bright versus CXCR4brightCD5dim cells and identified differential expression of dozens of molecules with a putative function cell migration. We have shown that GRB2 associated binding protein 1 (GAB1) positively regulates CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches is mediated by FoxO1-based transcriptional GAB1 activation. We also describe that upregulation of GAB1 plays an important role in maintaining basal PI3K activity and "tonic" AKT phosphorylation required to sustain survival of resting CLL cells. This is important during ibrutinib therapy since CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit "tonic" or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.
Background Cutaneous wounds in patients with diabetes exhibit impaired healing due to physiological impediments and conventional care options are severely limited. Multipotent stromal cells (MSCs) have been touted as a powerful new therapy for diabetic tissue repair owing to their trophic activity and low immunogenicity. However, variations in sources and access are limiting factors for broader adaptation and study of MSC-based therapies. Amniotic fluid presents a relatively unexplored source of MSCs and one with wide availability. Here, we investigate the potential of amniotic fluid-derived multipotent stromal cells (AFMSCs) to restore molecular integrity to diabetic wounds, amend pathology and promote wound healing. Method We obtained third trimester amniotic fluid from term cesarean delivery and isolated and expanded MSCs in vitro. We then generated 10 mm wounds in Leprdb/db diabetic mouse skin, and splinted them open to allow for humanized wound modeling. Immediately after wounding, we applied AFMSCs topically to the sites of injuries on diabetic mice, while media application only, defined as vehicle, served as controls. Post-treatment, we compared healing time and molecular and cellular events of AFMSC-treated, vehicle-treated, untreated diabetic, and non-diabetic wounds. A priori statistical analyses measures determined significance of the data. Result Average time to wound closure was approximately 19 days in AFMSC-treated diabetic wounds. This was significantly lower than the vehicle-treated diabetic wounds, which required on average 27.5 days to heal (p < 0.01), and most similar to time of closure in wild type untreated wounds (an average of around 18 days). In addition, AFMSC treatment induced changes in the profiles of macrophage polarizing cytokines, resulting in a change in macrophage composition in the diabetic wound bed. We found no evidence of AFMSC engraftment or biotherapy induced immune response. Conclusion Treatment of diabetic wounds using amniotic fluid-derived MSCs encourages cutaneous tissue repair through affecting inflammatory cell behavior in the wound site. Since vehicle-treated diabetic wounds did not demonstrate accelerated healing, we determined that AFMSCs were therapeutic through their paracrine activities. Future studies should be aimed towards validating our observations through further examination of the paracrine potential of AFMSCs. In addition, investigations concerning safety and efficacy of this therapy in clinical trials should be pursued.
Exosomes, nanosized extracellular vesicles, may be key to translating multipotent stromal cell (MSC) therapy to the bedside. We previously found that nuclear factor erythroid 2-related factor 2 (Nrf2) regulates MSC promotion of tissue repair in diabetes. Here, we explore a novel role of Nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects MSCs have on wound healing in mice with type 2 diabetes. Exosomes were harvested by differential ultracentrifugation of conditioned human bone marrow derived MSC media. For Nrf2-active exosomes, MSCs were incubated with potent Nrf2 activator, CDDO-Im. MSC characterization demonstrated robust tri-lineage differentiation, >95% expression of positive markers (CD44/CD73/CD90/CD105/CD106), and <5% expression of negative markers (CD45/CD31/CD14/CD19/HLA-DR). Immunoblotting of exosome isolates demonstrates enrichment for CD81, CD9 and TSG101. Nanoparticle tracking analysis demonstrates that Nrf2-active MSCs increase exosome secretion by 54%, compared to Nrf2-baseline MSCs (p<0.05). Exosomes were injected into the wound margin of full-thickness wounds on Leprdb/db (db/db) mice. Both Nrf2-baseline and Nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p<0.05). This reduction eliminated the delay in closure time compared to wounds of C57/B6 mice. Nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated C57/B6 wounds. At day 10, exosome treated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of CD31+ vessels compared to vehicle-treated wounds. We conclude that activating Nrf2 in MSCs increases isolation efficiency by amplifying exosome yield and content. MSC exosome based therapies have potential for rapid translation to improve clinical wound healing outcomes. Disclosure J. Kuhn: None. A. Hassan: None. S. Sharma: None. J. Kwong: None. M. Rahman: None. S. Adam: None. J. Lee: None. A.P. Villarreal Ponce: None. P.S. Rabbani: None. Funding Plastic Surgery Foundation; Wound Healing Society; New York University Clinical and Translational Science Institute
The synchronous activation of BCR and CD40 signaling via B-T cell interactions is required for proliferation of normal (Luo et al, 2018) and some malignant B cells, especially in chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). In CLL/FL cells, proliferation occurs mainly in lymph nodes, but not in bone marrow or peripheral blood which lack access to proper B-T cell interactions. Here we have analyzed the mRNA and miRNAs profile in the proliferative intraclonal CLL cell subpopulation that has recently exited lymph node niches (CXCR4dimCD5bright cells; Calissano et al,2011; Pavlasova et al,2016) to reveal molecules potentially participating in synchronous regulation of CD40 and BCR pathway. This has identified 36 miRNAs and 1370 mRNAs differentially expressed in CLL cells exiting lymph nodes as compared to resting non-proliferative CLL cells (CXCR4brightCD5dim cells). Next, we overlapped the 36 miRNAs with their predicted target mRNAs with putative function in CD40/BCR (TargetScan, KEGG) which were anti-correlated to at least one miRNA (miRNAs typically decrease mRNA stability). This revealed among others anti-correlation of lower miR-29 with higher TRAF4 levels in immune niches, which was validated in paired lymph node biopsies vs peripheral blood CLL cells (P<0.01). The negative regulation of TRAF4 by miR-29 was confirmed by transfection of primary CLL and MEC1 cells with synthetic miR-29, miR-29 inhibitor and by a luciferase assay with binding site from TRAF4 3'UTR. Several other miR-29 targets involved in other pathways have been identified as additional targets. TRAF4 is an understudied member of a protein family regulating receptor-induced immune cell activation, however, its role in CD40 pathway or its interactome is unknown. Co-immunoprecipitation and mass spectrometry in B cells pre- and post- stimulation by CD40 ligand revealed TRAF4 interaction with members of the CD40 pathway and a novel function in positive regulation of CD40-induced NFκB activity. In B cells, TRAF4 silencing or its over-expression affected IKKα/β phosphorylation following CD40 ligation or co-culture with activated T cells (all P<0.01). Notably, BCR inhibition lead in vivo to miR-29 upregulation by interfering with its negative regulator MYC induced by BCR. This leads to TRAF4 repression and drastically impairs CD40 signaling (P<0.001), which at least partially explains the observed anti-proliferative effects of BCR inhibitors. Altogether, we have described a novel MYC-miR-29-TRAF4 axis that regulates CD40 signaling in B cells, and acts to synchronize BCR activation with CD40 pathway. Supported by: The ERC under the European Union's Horizon 2020 research and innovation program (grant agreement No 802644), MH CZ, grant nr. NV18-03-00054 and NU20-03-00292. All rights reserved. Czech Science Foundation (20-02566S), MEYS CZ under the project CEITEC 2020 (LQ1601), MH CZ-DRO (FNBr,65269705) and MUNI/A/1595/2020. Citation Format: Sonali Sharma, Vaclav Seda, Eva Vojackova, Gabriela Mladonicka Pavlasova, Daniel Filip, Laura Ondrisova, Veronika Sandova, Lenka Kostalova, Pedro F. Zeni, Marek Borsky, Sarka Pospisilova, Medhat Shehata, Laura Z. Rassenti, Ulrich Jaeger, Michael Doubek, Matthew S. Davids, Jennifer R. Brown, Jiri Mayer, Thomas J. Kipps, Marek Mraz. miR-29-TRAF4 axis is a novel regulator of CD40 signaling in malignant B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2368.
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