Existence of reprogrammed lymphoma stem cells in a murine ALCL-like model.

Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN AlkatrasFunder: DFG (CRC850)Funder: DFG (CRC850) BMBF (DeCaRe, FKZ 01ZX1409B)Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN Alkatras DFG (FOR 2033 B1)Funder: DJCLS (R14/22) MSCA-ITN-2015-ETN Alkatras Grant from the Government of Baden-Württemberg (BSL)While cancer stem cells are well established in certain hematologic and solid malignancies, their existence in T cell lymphoma is unclear and the origin of disease is not fully understood. To examine the existence of lymphoma stem cells, we utilized a mouse model of anaplastic large cell lymphoma. Established NPM-ALK+ lymphomas contained heterogeneous cell populations ranging from mature T cells to undifferentiated hematopoietic stem cells. Interestingly, CD4-/CD8- double negative (DN) lymphoma cells aberrantly expressed the T cell receptor α/β chain. Serial transplantation of sorted CD4/CD8 and DN lymphoma subpopulations identified lymphoma stem cells within the DN3/DN4 T cell population, whereas all other subpopulations failed to establish serial lymphomas. Moreover, transplanted lymphoma DN3/DN4 T cells were able to differentiate and gave rise to mature lymphoma T cells. Gene expression analyses unmasked stem-cell-like transcriptional regulation of the identified lymphoma stem cell population. Furthermore, these lymphoma stem cells are characterized by low CD30 expression levels, which might contribute to limited long-term therapeutic success in patients treated with anti-CD30-targeted therapies. In summary, our results highlight the existence of a lymphoma stem cell population in a NPM-ALK-driven CD30+ mouse model, thereby giving the opportunity to test innovative treatment strategies developed to eradicate the origin of disease

Current Immunotherapeutic Approaches in T Cell Non-Hodgkin Lymphomas

T cell non-Hodgkin lymphoma (T-NHL) is a rare and heterogeneous group of neoplasms of the lymphoid system. With the exception of a few relatively indolent entities, T-NHL is typically aggressive, treatment resistant, and associated with poor prognosis. Relatively few options with proven clinical benefit are available for patients with relapsed or refractory disease. Immunotherapy has emerged as a promising treatment for the management of patients with hematological malignancies. The identification of tumor antigens has provided a large number of potential targets. Therefore, several monoclonal antibodies (alemtuzumab, SGN-30, brentuximab vedotin, and mogamulizumab), directed against tumor antigens, have been investigated in different subtypes of T-NHL. In addition to targeting antigens involved in cancer cell physiology, antibodies can stimulate immune effector functions or counteract immunosuppressive mechanisms. Chimeric antigen receptor (CAR)-T cells directed against CD30 and immune checkpoint inhibitors are currently being investigated in clinical trials. In this review, we summarize the currently available clinical evidence for immunotherapy in T-NHL, focusing on the results of clinical trials using first generation monoclonal antibodies, new immunotherapeutic agents, immune checkpoint inhibitors, and CAR-T cell therapies

Gab2 deficiency prevents Flt3-ITD driven acute myeloid leukemia in vivo

Internal tandem duplications (ITD) of the FMS-like tyrosine kinase 3 (FLT3) predict poor prognosis in acute myeloid leukemia (AML) and often co-exist with inactivating DNMT3A mutations. In vitro studies implicated Grb2-associated binder 2 (GAB2) as FLT3-ITD effector. Utilizing a Flt3-ITD knock-in, Dnmt3a haploinsufficient mouse model, we demonstrate that Gab2 is essential for the development of Flt3-ITD driven AML in vivo, as Gab2 deficient mice displayed prolonged survival, presented with attenuated liver and spleen pathology and reduced blast counts. Furthermore, leukemic bone marrow from Gab2 deficient mice exhibited reduced colony-forming unit capacity and increased FLT3 inhibitor sensitivity. Using transcriptomics, we identify the genes encoding for Axl and the Ret co-receptor Gfra2 as targets of the Flt3-ITD/Gab2/Stat5 axis. We propose a pathomechanism in which Gab2 increases signaling of these receptors by inducing their expression and by serving as downstream effector. Thereby, Gab2 promotes AML aggressiveness and drug resistance as it incorporates these receptor tyrosine kinases into the Flt3-ITD signaling network. Consequently, our data identify GAB2 as a promising biomarker and therapeutic target in human AML

Inhibition of Aurora Kinase B Is Important for Biologic Activity of the Dual Inhibitors of BCR-ABL and Aurora Kinases R763/AS703569 and PHA-739358 in BCR-ABL Transformed Cells

<div><p>ABL tyrosine kinase inhibitors (TKI) like Imatinib, Dasatinib and Nilotinib are the gold standard in conventional treatment of CML. However, the emergence of resistance remains a major problem. Alternative therapeutic strategies of ABL TKI-resistant CML are urgently needed. We asked whether dual inhibition of BCR-ABL and Aurora kinases A-C could overcome resistance mediated by ABL kinase mutations. We therefore tested the dual ABL and Aurora kinase inhibitors PHA-739358 and R763/AS703569 in Ba/F3- cells ectopically expressing wild type (wt) or TKI-resistant BCR-ABL mutants. We show that both compounds exhibited strong anti-proliferative and pro-apoptotic activity in ABL TKI resistant cell lines including cells expressing the strongly resistant T315I mutation. Cell cycle analysis indicated polyploidisation, a consequence of continued cell cycle progression in the absence of cell division by Aurora kinase inhibition. Experiments using drug resistant variants of Aurora B indicated that PHA-739358 acts on both, BCR-ABL and Aurora Kinase B, whereas Aurora kinase B inhibition might be sufficient for the anti-proliferative activity observed with R763/AS703569. Taken together, our data demonstrate that dual ABL and Aurora kinase inhibition might be used to overcome ABL TKI resistant CML.</p></div

Personalized Treatment Selection and Disease Monitoring Using Circulating Tumor DNA Profiling in Real-World Cancer Patient Management

BACKGROUND Circulating tumor DNA (ctDNA) in the blood plasma of cancer patients is an emerging biomarker used across oncology, facilitating noninvasive disease monitoring and genetic profiling at various disease milestones. Digital droplet PCR (ddPCR) technologies have demonstrated high sensitivity and specificity for robust ctDNA detection at relatively low costs. Yet, their value for ctDNA-based management of a broad population of cancer patients beyond clinical trials remains elusive. METHODS We developed mutation-specific ddPCR assays that were optimized for their use in real-world cancer management, covering 12 genetic aberrations in common cancer genes, such as EGFR, BRAF, KIT, KRAS, and NRAS. We assessed the limit of detection (LOD) and the limit of blank (LOB) for each assay and validated their performance for ctDNA detection using matched tumor sequencing. RESULTS We applied our custom ddPCR assays to 352 plasma samples from 96 patients with solid tumors. Mutation detection in plasma was highly concordant with tumor sequencing, demonstrating high sensitivity and specificity across all assays. In 20 cases, radiographic cancer progression was mirrored by an increase of ctDNA concentrations or the occurrence of novel mutations in plasma. Moreover, ctDNA profiling at diagnosis and during disease progression reflected personalized treatment selection through the identification of actionable gene targets in 20 cases. CONCLUSION Collectively, our work highlights the potential of ctDNA assessment by sensitive ddPCR for accurate disease monitoring, robust identification of resistance mutations, and upfront treatment selection in patients with solid tumors. We envision an increasing future role for ctDNA profiling within personalized cancer management in daily clinical routine

Expression of the resistant Aurora B G160V mutant partly rescues cell division failures after exposure to PHA-739358 and R763/AS703569.

<p>Ba/F3 p185 wt and Aur B G160V expressing Ba/F3 p185 wt cells were cultured in the presence of 500 nM PHA-739358 (A) or 50 nM R763/AS703569 (B), harvested at the indicated time points and analysed by flow cytometry to determine DNA content of propodium iodide (PI) stained cells.</p

Characterization of Aurora model system.

<p>Crystal structure was analyzed in order to identify residues, whose specific mutation should abolish inhibitor binding while keeping Aurora kinase activity. (A) Superimposition of the CK2 crystal structure (green) with Aurora A (pink) - PHA-739358 (yellow) complex crystal structure, showing the position of the point mutation L210 and G216. (B) <i>Xenpus laevis</i> Aurora B (green) and INCENP (turquoise) in complex with AS7035369, showing the position of the point mutation L154 and G160. (C) Ba/F3 p185 wt cells were transfected with Babe-puro based retrovirus encoding AurA L210M, AurA G216V, AurB L154M, or AurB G160V point mutations. Empty pBabe-vector was used as control. Selection was accomplished with puromycin. Western blot analysis showing flag-tagged Aurora A and B expression similar to endogenous Aurora A/B protein levels. (D) Amino acid sequence alignment of human Aurora kinase A and B. Mutated residues are framed. It is of note that the point mutations L210M and G216V in Aurora A accord with L154M and G160V in Aurora B.</p

Suppression of Aurora B kinase activity by PHA-739358 or R763/AS703569 inhibits cell division and induces apoptosis in BCR-ABL negative and positive Ba/F3 cells.

<p>Ba/F3 and Ba/F3 p185 cells comprising the T315I, Y253F, and F317L mutations were exposed to the indicated concentration of PHA-739358 (A) or R763/AS703569 (B). After 24 h analysis of DNA content and apoptotic fraction of PI-stained cells were assessed by flow cytometry. Untreated cells served as control. Apoptosis was measured as the percentage of cells of sub-G1 DNA content in the FL2 channel in a logarithmic scale.</p

PHA-739358 and R763/AS703569 reduce BCR-ABL kinase activity at comparable concentrations and independent of the BCR-ABL mutation status.

<p>Ba/F3 p185 wt and IM-resistant T315I, Y253F, and F317L mutants cells were exposed to increasing concentrations of PHA-739358 (A) or R763/AS703569 (B) for 2.5 h and assessed for phosphorylation status of BCR-ABL and its downstream target STAT5 by western blot analysis. Untreated and DMSO treated cells were used as a controls.</p

Aurora Kinase inhibitors PHA-739358 and R763/AS703569 compromise cell proliferation and viability at different concentrations in BCR-ABL negative and positive Ba/F3 cells independent of the BCR-ABL mutation status.

<p>(A) Dose-effect curves for treatment with the indicated concentrations of PHA-739358 and R763/AS703569 in Ba/F3 and Ba/F3 p185 cells, including the IM-resistant mutants T315I, Y253F and F317L, after 48 h. Effects on cell proliferation were assessed by MTT assay. Shown is one representative experiment of three experiments performed. The percentage of cell growth was normalized to the growth of untreated control cells. (B) Bar graph quantifying cell proliferation after exposure to 200 nM PHA-739358 or 15 nM R763/AS703569 for 48 h. Untreated and DMSO-treated cells were used as controls. Shown is one representative experiment of three experiments performed. Values are expressed as mean of triplicates ± SD. The difference between Ba/F3 p185 wt and BCR-ABL mutants is statistically significant. (C) IC₅₀ values of BCR-ABL positive and negative Ba/F3 cells, calculated from the results after 48 h of incubation with PHA-739358 or R763/AS703569.</p