Periodic Reporting for period 4 - LeukemiaEnviron (SIGNALING PROPENSITY IN THE MICROENVIRONMENT OF B CELL CHRONIC LYMPHOCYTIC LEUKEMIA)
Berichtszeitraum: 2023-12-01 bis 2024-05-31
B cell chronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults. CLL cells are characterized by their universal dependency on pro-survival and pro-proliferative signals from immune niches. We aimed to reveal how the malignant B cells change the propensity of their signaling pathways in response to the different microenvironments such as peripheral blood vs lymph node to obtain the proliferative signals. This is of major relevance for CLL, but also transferable to the biology of some other B cell malignancies and/or normal B cells. We focused on understanding the mechanisms of regulation of signaling propensity of two pathways responsible for the proliferation and survival of CLL cells, namely B Cell Receptor (BCR) signaling and signals from T-cells mediated by CD40/IL4. In aim 1 we discovered that CD20 is one of the key proteins involved in CLL cell activation and influences BCR and interleukin signaling. This has important therapeutic implications since CD20 has been used as a therapeutic target for 20 years (rituximab), but its function in CLL/normal B cells is unknown. We also described that during BCR inhibitor therapy CLL cells adapt and rewire the intracellular signaling and BCR pathway, including changes in levels of CD20 and pAKT. In aim 2, we described that miR-29 acts as a critical regulator of T-cell signaling and downstream cell activation. This represents the first example of miRNAs' role in the propensity of T-cell interaction and could also be utilized therapeutically. In aim 3, we integrated our microenvironmental signaling data and developed a co-culture model that mmicks CLL-T cell interactions. This allows a robust proliferation of CLL cells in vitro, and they can be transferred to a mouse model for PDX that allows stable engraftment of primary CLL cells. These are breakthrough tools since CLL cells do not divide spontaneously in vitro and do not permanently engraft in mice which complicates studies of its biology.
Importantly, the inhibition of both BCR signaling and CLL cells' re-circulation to lymph nodes where BCR activation and T cell interactions occur is a key mechanism of action of BCR inhibitors clinically approved to inhibit BCR-associated kinases BTK or PI3K. We have demonstrated that non-genetic adaptation allows CLL cell adaptation, ensuring their survival in the peripheral blood when BTK/PI3K inhibitors inhibit BCR signaling and re-circulation to immune niches. Our results have important implications for combinatorial therapy in CLL.
Importantly, the inhibition of both BCR signaling and CLL cells' re-circulation to lymph nodes where BCR activation and T cell interactions occur is a key mechanism of action of BCR inhibitors clinically approved to inhibit BCR-associated kinases BTK or PI3K. We have demonstrated that non-genetic adaptation allows CLL cell adaptation, ensuring their survival in the peripheral blood when BTK/PI3K inhibitors inhibit BCR signaling and re-circulation to immune niches. Our results have important implications for combinatorial therapy in CLL.
In aim 1, we showed that CD20 is one of the key proteins involved in BCR-mediated CLL cell activation. This also has important therapeutic implications since CD20 is used as a therapeutic target for 20 years, but its regulation and function remain largely unknown. We have described for the first time that T-cell interactions determine CD20 levels on malignant B cells, and such levels impact IL4 signaling in a feed-forward regulatory loop. We have also revealed that drugs targeting PI3K kinase will lead to reduced CD20 levels and, therefore, are less suitable for combination with anti-CD20 antibodies. In aim 1, we also described a mechanism that allows malignant B lymphocytes to survive therapy with so-called BCR inhibitors (ibrutinib/idelalisib). We have described two completely novel mechanisms. The first one involves the induction of GAB1 protein and subsequent increase in tonic pro-survival signaling mediated by AKT kinase. The second one involved the induction of the mTORC2 assembly protein Rictor via the transcription factor FoxO1, which activates Akt irrespective of cell surface receptors/BCR-associated kinases. We have further shown that GAB1 or FoxO1 inhibitors can kill leukemic cells and could be used alone or in combination with BTK or PI3K inhibitors.
In aim 2, we described the first example of the role of short non-coding RNAs, called microRNAs , in regulating CLL-T cell interactions. The described mechanism provides a mechanistic way for B cell to synchronously activate both BCR and CD40, which is required to enter B cells into the cell cycle and multiplication. We have proved the regulation of CD40 signaling by miR-29 in CLL and also in a related disease, follicular lymphoma. We also showed that the BCR inhibitors ibrutinib or idelalisib affect this axis, and the propensity of CD40 signaling also changes during the transformation of lymphomas into more aggressive disease. These data also explain why BCR inhibitors have such an unexpected and dramatic effect on the proliferation of malignant B cells.
In aim 3, we integrated our data on microenvironmental signaling and developed a novel CLL co-culture model that allows us to mimic CLL-T cell interactions and triggers robust CLL cell proliferation. We utilized the analyses of microenvironmental interactions to define what signals are missing in in vitro and in immunodeficient mice, and preclude the proliferation/engraftment of leukemic CLL cells. We genetically engineered supportive cells to express three T-cell factors. According to our data, this model is the most robust model for studying CLL cell proliferation, which is otherwise impossible since CLL cells in vitro do not spontaneously proliferate. Moreover, in line with our plan, we managed to transfer this model on 3D scaffold to a mouse model for patient-derived xenograft (PDX). This allows stable engraftment of primary CLL cells and studies of CLL biology. The co-culture model allowed us to also reveal for the first time that pan-RAF inhibitors are able to block CLL cells proliferation.
In aim 2, we described the first example of the role of short non-coding RNAs, called microRNAs , in regulating CLL-T cell interactions. The described mechanism provides a mechanistic way for B cell to synchronously activate both BCR and CD40, which is required to enter B cells into the cell cycle and multiplication. We have proved the regulation of CD40 signaling by miR-29 in CLL and also in a related disease, follicular lymphoma. We also showed that the BCR inhibitors ibrutinib or idelalisib affect this axis, and the propensity of CD40 signaling also changes during the transformation of lymphomas into more aggressive disease. These data also explain why BCR inhibitors have such an unexpected and dramatic effect on the proliferation of malignant B cells.
In aim 3, we integrated our data on microenvironmental signaling and developed a novel CLL co-culture model that allows us to mimic CLL-T cell interactions and triggers robust CLL cell proliferation. We utilized the analyses of microenvironmental interactions to define what signals are missing in in vitro and in immunodeficient mice, and preclude the proliferation/engraftment of leukemic CLL cells. We genetically engineered supportive cells to express three T-cell factors. According to our data, this model is the most robust model for studying CLL cell proliferation, which is otherwise impossible since CLL cells in vitro do not spontaneously proliferate. Moreover, in line with our plan, we managed to transfer this model on 3D scaffold to a mouse model for patient-derived xenograft (PDX). This allows stable engraftment of primary CLL cells and studies of CLL biology. The co-culture model allowed us to also reveal for the first time that pan-RAF inhibitors are able to block CLL cells proliferation.
We have made substantial discoveries in the field of microenvironmental interactions of malignant B cells, primarily CLL. We have demonstrated for the first time microRNAs (miRNA) and other non-coding RNAs play a key role in regulating interactions in the microenvironment. In addition, we described the regulation and function of CD20, as this molecule has been used as a therapeutic target (rituximab, obinutuzumab) for more than 20 years, but its regulation and function was unclear. We believe that a better understanding of CD20 and non-coding RNAs can lead to novel drugs (such as miRNA-inhibitors) or improved anti-CD20 targeting in combination with other drugs. The approval of drugs that inhibit B-cell receptor (BCR) associated kinases and target interactions in the microenvironment was a milestone in the therapy of B cell neoplasms. During the ERC project, we described novel pathways activated in the microenvironment and affected by BCR inhibition and their relationship to leukemia cells' survival mechanisms. The understanding the regulation of microenvironment interactions and adaptation to targeted therapy allowed the identification of new prognostic and predictive biomarkers in hematological malignancies, and we have provided pre-clinical evidence that 2 novel inhibitors can be used alone or together with BCR inhibitors. We also developed a novel tool to study the proliferation of CLL cells utilizing genetically engineered cells that mimic C-T cell interactions in vitro and in a novel PDX model (based on co-transplantation of modified stromal cells on 3D scaffold with CLL cells). This tool also allowed us to discover the outstanding potency of pan-RAF inhibitors for blocking CLL proliferation. Altogether, we successfully fulfilled all the main objectives of the grant and published the results in high-profile hematological journals.