Lesca-Miriam Holdt

Acute myeloid leukemia (AML) is a highly aggressive malignancy with poor prognosis, underlining the need for suitable model systems to develop novel treatments. To complement the few existing AML cell lines and restricted primografts, we transplanted more than 100 primary AML samples into immunocompromised mice and generated 23 exceptionally robust patient‐derived xenograft (*PDX) models of AML that allow virtually unlimited serial transplantation and efficient genetic engineering. These models represent individuals of all age groups and several WHO subgroups. *PDX models substantially outperform AML cell lines in preserving leukemia biology and include AML subgroups for which no cell lines exist, such as cytogenetically normal or IDH1/2 ‐mutant AML or simultaneous NPM1 mutation and FLT3‐ ITD. Their resilience to freeze‐thaw cycles supports broad dissemination across research institutions through sharing via CancerModels.org. Using lentiviruses, we stably expressed luciferase for longitudinal, noninvasive, and sensitive real‐time monitoring of leukemia burden and therapeutic response in vivo . This approach enabled rigorously controlled preclinical studies, including phase II‐like trials, which demonstrated highly variable treatment responses between different *PDX models, mimicking the heterogeneity in patient cohorts. Long‐term treatment, including repeated cytarabine exposure over 1 year, showed a yet undescribed decrease in leukemia's proliferation rate. These insights underscore the platform's distinctive capacity to study treatment dynamics over clinically relevant timeframes. Collectively, our unique resource represents a powerful, robust, and versatile platform with strong potential to accelerate translational AML research for the ultimate benefit of patients.

MALT1 (mucosa-associated lymphoid tissue lymphoma/leukemia protein 1)–TRAF6 [tumor necrosis factor receptor (TNFR)-associated factor 6] interaction drives lymphocyte activation and adaptive immunity, but it also contributes to maintaining immune homeostasis. MALT1 exists in two isoforms that differ only by either encoding two (MALT1A) or one (MALT1B) TRAF6 binding motif (T6BM). The human mutation MALT1 E806D in T6BM2, expressed in both MALT1A and MALT1B, has been associated with an immune disorder combining symptoms of immune deficiency and autoimmunity. Here, we report that the orthologous germline mutation MALT1 E814D is sufficient to induce a fatal autoimmune syndrome in mice. We demonstrate that species-specific differences in the effects of T6BM2 disruptions can be attributed to alterations in MALT1 splicing and that immune homeostasis is restored by genetically enforcing expression of MALT1A in MALT1 E814D mice. Thus, alternative MALT1 splicing allows tuning of TRAF6 association, thereby functioning as a molecular rheostat to balance between optimal immune activation and maintenance of peripheral tolerance.

Preclinical cancer research requires robust model systems, especially for poor prognosis entities like acute myeloid leukemia (AML), a highly aggressive blood cancer. Here, primary tumor cells from 137 AML patients of all age groups were transplanted into immune compromised mice to generate patient-derived xenografts (PDX). From these, 23 models enable robust, virtually endless serial re-transplantation and are amenable to lentiviral genetic engineering ( ^* PDX AML models). These models primarily originate from patients with highly aggressive, relapsed disease. Comprehensive genomic, transcriptomic, and epigenomic analyses confirmed that they replicate primary AML biology more faithfully than conventional cell lines. Notably, ^* PDX AML models include AML subgroups that are underrepresented or absent in existing model systems, such as cytogenetically normal or IDH1/2 -mutant AML. They withstand freeze-thaw cycles, making them suitable for broad distribution and reproducibility across research institutions. Luciferase-based in vivo imaging enables real-time monitoring of tumor progression and treatment responses in preclinical trials. Surprisingly, long-term treatment, including repeated cytarabine therapy over a period of one year, showed a gradual reduction in leukemia cell proliferation, which decreased continuously after each treatment block. Collectively, our ^* PDX models represent a robust, versatile, and relevant platform that holds great promise to accelerate translational research for the benefit of cancer patients. Visual Key Points: We present new robust AML PDX models covering subgroups for which no cell lines exist for use in various ex vivo and in vivo applications. ^* PDX models enable serial transplantation, genetic engineering and better representation of primary AML biology than cell lines. One-year in vivo trials mimicking clinical chemotherapy showed surprising gradual decline in leukemia growth after each treatment block.

Context: Milk protein contains high concentrations of branched-chain amino acids (BCAA) that play a critical role in anabolism and are implicated in the onset of obesity and chronic disease. Characterizing BCAA catabolism in the postprandial phase could elucidate the impact of protein intake on obesity risk established in the “early protein hypothesis.” Objective: To examine the acute effects of protein content of young child formulas as test meals on BCAA catabolism, observing postprandial plasma concentrations of BCAA in relation to their degradation products. Methods: The TOMI Add-On Study is a randomized, double-blind crossover study in which 27 healthy adults consumed 2 isocaloric young child formulas with alternating higher (HP) and lower (LP) protein and fat content as test meals during separate interventions, while 9 blood samples were obtained over 5 hours. BCAA, branched-chain α-keto acids (BCKA), and acylcarnitines were analyzed using a fully targeted HPLC-ESI-MS/MS approach. Results: Mean concentrations of BCAA, BCKA, and acylcarnitines were significantly higher after HP than LP over the 5 postprandial hours, except for the BCKA α-ketoisovalerate (KIVA). The latter metabolite showed higher postprandial concentrations after LP. With increasing mean concentrations of BCAA, concentrations of corresponding BCKA, acylcarnitines, and urea increased until a breakpoint was reached, after which concentrations of degradation products decreased (for all metabolites except valine and KIVA and Carn C4:0-iso). Conclusion: BCAA catabolism is markedly influenced by protein content of the test meal. We present novel evidence for the apparent saturation of the BCAA degradation pathway in the acute postprandial phase up to 5 hours after consumption.

To date, responsiveness to tumor necrosis factor alpha inhibitors in ulcerative colitis (UC) patients is not predictable. This is partially due to a lack of understanding of the underlying inflammatory processes. The aim of this study was to identify immunological subgroups of patients with UC and to test responsiveness to adalimumab in these subgroups in the mouse model of ulcerative colitis (UC), which is based on NOD/scid IL-2Rγ ^null (NSG) mice reconstituted with peripheral blood mononuclear cells (PBMCs; NSG-UC). The immunological profiles of 40 UC patients and 16 non-UC donors were determined by flow cytometric analysis of PBMCs in a snapshot and longitudinal study and analyzed by principal component, orthogonal partial least square discrimination (oPLS-DA), and hierarchical clustering analysis. NSG mice were reconstituted 5 times at consecutive time points with PBMCs from a single donor and were analyzed for frequencies of human leukocytes and histological phenotype. The response to adalimumab of 2 identified subgroups was tested in the NSG-UC model. We used the clinical, colon, and histological score, serum levels of glutamic and aspartic acid, and IL-6 and IL-1ß. Response was analyzed by oPLS-DA. Analysis revealed a distinction between UC and non-UC donors. Hierarchical clustering identified 2 major subgroups in UC patients. Group I was characterized by TH17 and M1 monocytes, group II by TH2/TH1, and switched B cells. These subgroups reflect the dynamics of inflammation as patients. NSG-UC mice achieved an immunological phenotype reflecting the patient’s immunological phenotype. oPLS-DA revealed that NSG-UC mice reconstituted with PBMCs from group II responded better to adalimumab. The combination of profiling and testing of therapeutics in the NSG-UC model may lead to individualized and phase-dependent therapies.

The aim of the current study was to investigate the cAMP‐dependent regulation of arginase‐1 (ARG1) expression in RAW‐macrophages. Basal ARG1 mRNA expression was low and increased upon incubation with the cAMP analogue Br‐cAMP. We used selective agonists of protein kinase A type I (PKAI), type II (PKAII) and exchange protein directly activated by cAMP (EPAC) to determine the pathway responsible for ARG1 expression. Activation of PKAI led to a significant up‐regulation of ARG1 mRNA expression and arginase enzyme activity. In contrast, neither activation of PKAII nor activation of EPAC affected ARG1 expression. In addition, it has been shown that histone deacetylase (HDAC) activity plays a critical role in cAMP‐dependent transcriptional regulation. Incubation with Br‐cAMP and the HDAC inhibitor trichostatin A (TSA) led to a concentration‐dependent suppression of ARG1 expression. These data indicate that cAMP‐dependent activation of ARG1 expression is mediated by PKAI and requires histone deacetylation. J. Cell. Biochem. 103: 520–527, 2008.