Checkpoint kinases sense replicative stress to prevent DNA damage. Here we show that the histone deacetylases HDAC1/HDAC2 sustain the phosphorylation of the checkpoint kinases ATM, CHK1 and CHK2, activity of the cell cycle gatekeeper kinases WEE1 and CDK1, and induction of the tumour suppressor p53 in response to stalled DNA replication. Consequently, HDAC inhibition upon replicative stress promotes mitotic catastrophe. Mechanistically, HDAC1 and HDAC2 suppress the expression of PPP2R3A/PR130, a regulatory subunit of the trimeric serine/threonine phosphatase 2 (PP2A). Genetic elimination of PR130 reveals that PR130 promotes dephosphorylation of ATM by PP2A. Moreover, the ablation of PR130 slows G1/S phase transition and increases the levels of phosphorylated CHK1, replication protein A foci and DNA damage upon replicative stress. Accordingly, stressed PR130 null cells are very susceptible to HDAC inhibition, which abrogates the S phase checkpoint, induces apoptosis and reduces the homologous recombination protein RAD51. Thus, PR130 controls cell fate decisions upon replicative stress.

The IL-7/IL-7R pathway is essential for lymphocyte development and disturbances in the pathway can lead to immune deficiency or T cell mediated destruction. Here, the effect of transient hyperexpression of IL-7 was investigated on immune regulation and allograft rejection under immunosuppression. An experimental in vivo immunosuppressive mouse model of IL-7 hyperexpression was developed using transgenic mice (C57BL/6 background) carrying a tetracycline inducible IL-7 expression cassette, which allowed the temporally controlled induction of IL-7 hyperexpression by Dexamethasone and Doxycycline treatment. Upon induction of IL-7, the B220 + c-kit + Pro/Pre-B I compartment in the bone marrow increased as compared to control mice in a serum IL-7 concentration-correlated manner. IL-7 hyperexpression also preferentially increased the population size of memory CD8 + T cells in secondary lymphoid organs, and reduced the proportion of CD4 + Foxp3 + T regulatory cells. Of relevance to disease, conventional CD4 + T cells from an IL-7-rich milieu escaped T regulatory cell-mediated suppression in vitro and in a model of autoimmune diabetes in vivo . These findings were validated using an IL-7/anti-IL7 complex treatment mouse model to create an IL-7 rich environment. To study the effect of IL-7 on islet graft survival in a mismatched allograft model, BALB/c mice were rendered diabetic by streptozotocin und transplanted with IL-7-inducible or control islets from C57BL/6 mice. As expected, Dexamethasone and Doxycycline treatment prolonged graft median survival as compared to the untreated control group in this transplantation mouse model. However, upon induction of local IL-7 hyperexpression in the transplanted islets, graft survival time was decreased and this was accompanied by an increased CD4 + and CD8 + T cell infiltration in the islets. Altogether, the findings show that transient elevations of IL-7 can impair immune regulation and lead to graft loss also under immune suppression.

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Background: Conventional CAR-T cells targeting CD123 in rrAML have achieved objective responses, but led to long-lasting myelosuppression due to expression of CD123 on progenitor cells. UniCAR-T-CD123 is a rapidly switchable two-component CAR-T therapy. An inert universal CAR-T cell (UniCAR-T) is combined with a CD123-specific soluble targeting module with a short half-life (TM123). By administering or withholding the continuous infusion of TM123, the UniCAR-T-cell can be rapidly switched on and off. Within the ongoing Phase IA study in rrAML, we investigated the expansion kinetics of UniCAR-T cells during TM123 administration in peripheral blood and bone marrow as well as cytokine profiles of treated patients. Methods: Prior to administration of autologous UniCAR-T cells, patients received a standard Flu/Cy lymphodepletion regimen at day -5 to -3. TM123 was administered as continuous infusion over 24 days starting at day 0. At day 1 a single dose of UniCAR-T cells was given. Dosing started with 1 x 108 UniCAR-T cells and 0.5 mg TM123 per day in patient 1. Patient 2 received the same TM123 dose and a UniCAR-T dose of 2.5 x 108 cells. Patient 3 received the same cell dose as patient 2 but a higher TM123 dose (1 mg/day). Pharmacokinetic of UniCAR-T and TM123 was determined from peripheral blood and bone marrow by droplet digital PCR and TM123-specific ELISA, respectively. Cytokine levels were measured by microfluidic immunoassay. Results: All 3 patients treated so far achieved an objective response, with one showing a PR and two a CRi. Treatment proved to be tolerable, no DLTs were observed to date and adverse events were mild. Grade 1 CRS (fever) was observed in 2 patients but subsided within 48 h after use of antipyretics. Myelosuppression was observed starting after lymphodepletion, which immediately recovered after TM123 withdrawal on day 24 in all patients, providing evidence for the rapid off-switch of UniCAR-T cells post TM123 administration. UniCAR-T cells expanded in all patients in peripheral blood and bone marrow comparable to data reported for conventional CD123 CAR-T products, and were so far detectable for up to 6 months after administration. Expansion kinetics were TM123-dependent. Patients showed periods of transient increase of IL-6, IFN-γ and TNF-α levels preceding peak expansion and decreasing after termination of TM123 administration. One patient showed additional coinciding momentary elevation of GM-CSF and IL-2. Conclusions: The initial clinical and translational results of UniCAR-T-CD123 represent, to our understanding, a first time evidence for a well-tolerated and effective rapidly switchable CAR-T product. Even though the number of patients treated so far is limited, the data obtained provide clinical proof-of-concept for the opportunity to abrogate side effects by withdrawal of TM123. Enrollment into the Phase IA study is ongoing. Citation Format: Armin Ehninger, Julia Riewaldt, Cordula Gründer, Kristin Franke, Maria Schreiber, Martin Wermke, Sabrina Kraus, Carla Kreissig, Jan Koedam, Michael Pehl, Gerhard Ehninger, Marc Cartellieri. Expansion kinetics and cytokine profiles of UniCAR-T-CD123, a rapidly switchable two-component CAR-T therapy, in patients with relapsed/refractory AML [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 1506.