Sebastian Schlaweck

Recurrent genetic and chromosomal aberrations drive multiple myeloma (MM) pathogenesis. Among these, the t(4;14) translocation leads to overexpression of fibroblast growth factor receptor 3 (FGFR3) and is associated with poor prognosis. However, therapeutic approaches directly targeting FGFR3-driven myeloma progression remain limited. Here, we investigated the single-agent activity of ponatinib, a multikinase inhibitor, in MM. KMS18 and U266 myeloma cell lines were treated with ponatinib, and apoptosis induction, as well as VEGF and IL-6 secretion, was assessed. RNA sequencing of MM cells revealed pathway alterations induced by ponatinib treatment, which were subsequently validated by Western blot analysis. In vivo, mice inoculated with 5T33 myeloma cells received ponatinib, and survival was monitored. Notably, ponatinib exerted potent single-agent antimyeloma activity in an FGFR3-dependent manner by inducing apoptosis and suppressing VEGF and IL-6 secretion through inhibition of JAK/STAT, PI3K/AKT, and MAPK signaling. In vivo administration prolonged survival in myeloma-bearing mice. Collectively, our findings demonstrate the therapeutic efficacy of ponatinib in FGFR3-expressing MM beyond selective FGFR3 inhibition, suggesting that concurrent suppression of multiple signaling pathways is a critical mechanism of action. These results highlight the therapeutic potential of combined FGFR3-targeted strategies in multiple myeloma and provide a rationale for further clinical investigation.

Background: FMS-like tyrosine kinase 3 (FLT3) mutations or internal tandem duplication occur in 30% of acute myeloid leukemia (AML) cases. In these cases, FLT3 inhibitors (FLT3i) are approved for induction treatment and relapse. Allogeneic hematopoietic stem cell transplantation (alloHSCT) remains the recommended post-induction therapy for suitable patients. However, the role of FLT3i therapy after alloHSCT remains unclear. Therefore, we investigated the three currently available FLT3i, gilteritinib, midostaurin, and quizartinib, in terms of their immunosuppressive effect on dendritic cells (DCs). DCs are professional antigen-presenting cells inducing T-cell responses to infectious stimuli. Highly activated DCs can also cause complications after alloHSCT, such as triggering Graft versus Host disease, a serious and potentially life-threatening complication after alloHSCT. Methods: To study the immunomodulatory effects on DCs, we differentiated murine and human DCs in the presence of FLT3i and performed immunophenotyping by flow cytometry and cytokine measurements and investigated gene and protein expression. Results: We detected a dose-dependent immunosuppressive effect of midostaurin, which decreased the expression of costimulatory markers like CD86, and found a reduced secretion of pro-inflammatory cytokines such as IL-12, TNFα, and IL-6. Mechanistically, we show that midostaurin inhibits TLR and TNF signaling and NFκB, PI3K, and MAPK pathways. The immunosuppressive effect of gilteritinib was less pronounced, while quizartinib did not show truncation of relevant signaling pathways. Conclusions: Our results suggest different immunosuppressive effects of these three FLT3i and may, therefore, provide an additional rationale for optimal maintenance therapy after alloHSCT of FLT3-positive AML patients to prevent infectious complications and GvHD mediated by DCs.

Acute-Graft- versus -Host disease (aGvHD) is a life-threatening complication after allogeneic stem-cell-transplantation. It is mediated by alloreactive T cells whose trafficking to aGvHD target organs is orchestrated by chemokines. We here asked whether CCL17 and its corresponding receptor CCR4 are involved in aGvHD development and severity. We applied an experimental mouse model of aGvHD in CCR4/CCL17 knockout mice and analyzed gut biopsies of GvHD patients. We show that the absence of CCR4 in transplanted T cells induced significantly less severe aGvHD. This was accompanied by reduced expression of Gata3. Mechanistically, only CD4 ⁺ , but not CD8 ⁺ CCR4 ^-/- T cells protected from aGvHD. We next identified dendritic cells in the small intestine to produce CCL17, which selectively recruited CD4 ⁺ T cells. IL-4 production by intestinal CD4 ⁺ T cells promoted proliferation of CD8 ⁺ T cells. In line, we detected an upregulation of CCL17 and Gata3 in human aGvHD samples. Our results indicate that local CCL17 production in aGvHD target organs recruits T cells, reinforcing local tissue damage and immune cell recruitment. We identified the JAK1/2-inhibitor ruxolitinib to dampen CCL17-expression, thereby reducing GvHD severity. We here dissect a to date unknown role of the CCL17-CCR4 axis in aGvHD, which might help to develop novel therapeutic strategies.

Sinusoidal obstruction syndrome (SOS) is a rare complication after allogeneic hematopoietic stem cell transplantation (alloHSCT) caused by endothelial dysfunction. Previous definitions and diagnostic criteria for the presence of SOS include bilirubinemia, hepatomegaly and weight gain, but histological evaluation is still the only way to prove the diagnosis of SOS. However, biopsy remains an invasive technique and is therefore undesirable in the alloHSCT scenario. Hence, a non-invasive diagnostic strategy is critical. Besides thorough clinical assessment and laboratory values, ultrasound examination remains part of the diagnostic workflow in clinical routine. Previous studies defined sonographic abnormalities, which are associated with the occurrence of SOS, but a standardized protocol to perform reliable abdominal ultrasound has not been finally defined. In this study, we evaluated a multi-parameter protocol including laboratory values as well as ultrasound examination pre- and post-alloHSCT. The application of this protocol was feasible in clinical practice and achieved a high inter- and intra-rater reliability. In our population, no case of SOS was identifiable and, in line with previous studies, no changes known to be associated with SOS were detected by ultrasound examination in our cohort. Additionally, we investigated subgroups of patients partly fulfilling SOS diagnostic criteria analyzing correlations between the fulfilled criteria and aberrances in ultrasound measurements pre- and post-alloHSCT. Although statistical examination may be limited by a small sample size and missing SOS cases, hyperbilirubinemia, thrombocytopenia and weight gain showed only a coincidence with selected, enlarged liver dimensions in few patients. This may underline the fact that hepatomegaly occurs as an unspecific finding after alloHSCT. Our protocol, including the ultrasound examination pre- and post-alloHSCT and laboratory parameters, may help to rule out SOS early, but validation in a greater population and different transplantation centers is required to warrant broader appliance. Nevertheless, we aim to contribute to an elaborate and standardized work-flow in peri-alloHSCT patient care.

The PI3Kδ-inhibitor Idelalisib is approved for the treatment of Non-Hodgkin lymphoma. However, its use has been decreased within the last years due to deleterious infections such as cytomegalovirus and pneumocystis jirovecii. Here, we have investigated the effect of Idelalisib on human monocyte-derived dendritic cells (DCs) as important players in the induction of immune responses. We found that Idelalisib-treated DCs displayed impaired T cell stimulatory function. PI3Kδ inhibition during differentiation resulted in decreased Interleukin-12, Interleukin-13 and TNFα production by DCs after lipopolysaccharide stimulation. Moreover, DCs showed decreased expression of the activation marker CD83 after Idelalisib treatment. Further, in line with this was the failure of Idelalisib-treated DCs to properly induce allogeneic T cells in a dose-dependent manner. Finally, activation of the NFκB pathway was also ablated in Idelalisib-treated DCs. Our results implicate that severe infectious complications may not only result from direct PI3Kδ-inhibition in T cells, but also from impaired DC function in Idelalisib-treated patients. Here, we provide new insight into the pathogenesis of Idelalisib-associated infectious complications. Our study may further provide a rationale for the use of Idelalisib as a novel therapeutic option in inflammatory diseases.