Teresa Poggio

Immunotherapy has revolutionized the treatment of solid cancers in recent years. However, T-cell non-Hodgkin lymphomas (T-NHL) originate from immune cells themselves and are biologically heterogeneous, rendering investigations of immune checkpoint inhibitor (ICI) mechanisms of action complex. Although case reports and individual anaplastic large cell lymphoma (ALCL) patients enrolled in T-NHL trials demonstrated favorable responses to ICIs, hyperprogression was observed in other T-NHL subtypes. We therefore used a syngeneic mouse model of anaplastic lymphoma kinase positive (ALK+) ALCL to investigate immune surveillance and ICI-induced immune response. Transplantation experiments combined with the depletion of relevant immune axes revealed that ALCL immune surveillance is mediated by CD4+ T cells and natural killer cells. Innate and adaptive immune cell infiltration was confirmed in a large series of primary human ALK+ ALCL samples. ICI monotherapy demonstrated robust efficacy in murine ALCL, inducing complete remissions in ∼50% of treated animals. Mechanistically, PD-L1 blockade reversed regulatory T cell-mediated immunosuppression and increased the frequency of circulating effector CD8+ T lymphocytes, thereby significantly prolonging survival. Importantly, CD4+ T cells proved indispensable for driving and sustaining immunotherapy-induced antitumor responses in murine ALCL. CD4+ T cells of nonresponder animals exhibited an exhausted phenotype and a transcriptomic T helper 22–like signature, implicating persistent T-cell exhaustion and polarization as a meaningful immune evasion mechanism. Our findings uncover CD4+ T cells as key players in spontaneous and immunotherapy-mediated anti–T-cell lymphoma immunity, which demonstrates the critically needed preclinical proof-of-concept for the safe and effective use of immunotherapy for ALCL.

Ruxolitinib is a potent JAK1/JAK2 inhibitor, approved for the treatment of primary myelofibrosis (PMF) patients based on the concept of inhibition of oncogenic signaling. However, the effect of ruxolitinib on JAK2-V617F allelic burden is modest, suggesting that inhibition of JAK2-V617F signaling-driven clone expansion is not the main mechanism of action. We evaluate whether ruxolitinib mainly blocks the proliferation of the malignant clone or exerts its effects also by targeting non-malignant cells. Therefore, we develop two JAK2-V617F-driven myeloproliferative neoplasm (MPN) mouse models harboring ruxolitinib resistance mutations. Mice carrying ruxolitinib-resistant JAK2-V617F-driven MPN respond to ruxolitinib treatment similar to mice with ruxolitinib-sensitive JAK2-V617F MPN with respect to reduction of spleen size, leukocyte count and pro-inflammatory cytokines in the serum. Ruxolitinib reduces pro-inflammatory cytokines in both stromal cells and non-malignant hematopoietic cells. Using a rigorous ruxolitinib resistance mutation approach, we can prove that ruxolitinib acts independent of oncogenic JAK2-V617F signaling and reduces the main features of MPN disease such as spleen size and leukocyte counts. Our findings characterize the mechanism of action for ruxolitinib in MPN.

The t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre–B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

Internal tandem duplications (ITD) of the FMS-like tyrosine kinase 3 (FLT3) frequently co-exist with inactivating DNMT3A mutations and predict poor prognosis in acute myeloid leukemia (AML). In vitro studies implicated the docking protein Grb2-associated binder 2 (GAB2) as FLT3-ITD effector. By 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 survived significantly longer, had reduced blast counts and exhibited an attenuated spleen and liver pathology as compared to their Gab2-proficient littermates. Further, leukemic bone marrow isolated from Gab2 deficient mice displayed a reduced colony forming unit capacity and an increased sensitivity towards the Flt3 inhibitor quizartinib. Using RNA sequencing, 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 as it induces their expression and additionally serves as a downstream effector. Consequently, Gab2 promotes AML aggressiveness as well as drug resistance by incorporating the receptor tyrosine kinases Axl and Ret into the Flt3-ITD signaling network. Therefore, our data identify GAB2 as a promising biomarker as well as a therapeutic target in human AML. Citation Format: Corinna Spohr, Teresa Poggio, Geoffroy Andrieux, Katharina Schönberger, Nina Cabezas-Wallscheid, Melanie Börries, Sebastian Halbach, Anna L. Illert, Tilman Brummer. Gab2 deficiency protects against Flt3-ITD driven acute myeloid leukemia in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 945.

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.

Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome-type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.