Project Lines (PL) and SFB-Network

PL-A. Identification of genetic and epigenetic factors contributing to MPN development and progression through an integrated genome-wide analysis of copy number variations, point mutations (deep exome-sequencing), DNA methylation-patterns, and gene expression.
Data obtained in F47 as well as in other studies suggest that the genetic background, epigenetic features, and somatic lesions (aberration networks) all contribute essentially to the manifestation, evolution, and progression of MPN. PL-A further developed this concept during the second SFB period by studying genetic, epigenetic, and somatic lesions in an integrative approach that took multiple factors and features as well as sub-clone formation in MPN into account. The two CF-PF of the SFB supported these efforts and cooperated closely with #02, #04, and #10 in this PL. Germline variations and gender-related aspects of MPN were studied in project #02, #04, and #09, and new somatic lesions and epigenetic patterns and targets in #02, #04, and #10. Sequencing studies focused on genes involved in the regulation of growth, survival, and distribution of LSC in MPN.

PL-B. Functional analysis of MPN development and progression: cellular hierarchies and MPN sub-clone formation, LSC-niche interactions, and the cytokine network.
During both periods, PL-B addressed the cellular complexity in various MPN to establish markers and tools to characterize and purify LSC, to examine niche cells and LSC-niche interactions, and to detect novel targets and signaling nodes (link to PL-A and PL-C) in these cells; #04, #06, #10, and #11 as well as the two CF-PF closely collaborated to reach these aims in the second SFB period. In CML, PMF, and MCL, the marker profile of LSC and assays to study LSC-niche interactions in vitro have been established. In patients with ET and PV, #04 screened for novel specific SC markers in the second funding period. Throughout the entire period PL-B studied cytokine networks regulating i) growth, adhesion, differentiation, and function of LSC and ii) expansion, remodeling, and activation of the SC microenvironment (e.g. angiogenic or fibrogenic cytokines). In addition, PL-B studied cytokines and cytokine-networks suppressing immune responses by upregulating certain recognition-receptors, like CD47, or immune checkpoint receptors, like PD-L1 (CD274) on LSC. PL-B also explored the cellular source of cytokines, with special emphasis on neoplastic cells. In addition, PL-B studied LSC sub-clone formation and the trans-differentiation capacity of LSC (#04). Sub-clone-evolution and expansion were analyzed in patients (follow-up samples), in various cell line models (co-culture-assays), and in in vivo xenotransplantation models using specific molecular markers and sequencing analyses. The trans-differentiation capacity of LSC was determined by analyzing clonal relationships between niche cells and LSC and by testing the biochemical basis of trans-differentiation in patient-derived iPSC-like and LSC-like cells. A specific aim was to learn how to block trans-differentiation in LSC by applying targeted drugs.

PL-C. Identification of major signaling molecules and networks as well as key effector molecules involved in the regulation of growth, survival, and drug resistance in MPN (stem) cells.
Throughout both periods, PL-C focused on 4 types of molecules: i) kinase drivers promoting pro-oncogenic signaling in neoplastic (stem) cells, ii) downstream signaling molecules that trigger survival and proliferation or drug resistance in LSC, iii) downstream effector molecules (cytokines, chemokines) involved in the regulation of growth of LSC and LSC-niche interactions and thereby trigger disease pathogenesis and resistance, and iv) epigenetically relevant molecules and transcription factors. These molecules were identified in MPN (stem) cells in #02 through #11 in collaboration with the biobank and omics CF-PF of our SFB. Signaling molecules or effector molecules were identified in human MPN cells and were then validated in appropriate mouse models. Expression and function of these molecules were confirmed for primary patient-derived MPN cells, LSC, or BM-derived niche cells (e.g. BM endothelial cells). The effects of potential autocrine cytokines on MPN cells and LSC was explored using inhibitory antibodies, shRNA or CRISPR/Cas9, or drugs directed against ligand-proteins or receptors. The impact of various signaling molecules was determined by using shRNA, CRISPR/Cas9, as well as specific chemical compounds. One critical aim in PL-C was the identification and characterization of cooperating signaling nodes and networks and of cooperating or amplifying networks of effector molecules mediating drug resistance in MPN (stem) cells. A number of different technologies and assays, including genome-wide analyses, synthetic lethality studies, shRNA/siRNA combination studies, drug-combination studies, and pharmacoscopy were applied to address this objective in the second SFB period. This was followed by in-depth analyses using most relevant targets and most promising drugs. Likewise, after having characterized BRD4 as a major target in AML and CML and JQ1 as suitable drug that blocks BRD4 activity in various myeloid neoplasms project #10 also revealed that neoplastic cells in advanced CML and several AML subsets are resistant to BET inhibition, and subsequently revealed mechanisms and genes contributing to resistance.

PL-D. Identification of new therapeutic targets and target-networks in MPN cells and translation of new treatment approaches, including LSC-eradicating drug combinations.
In this PL, SFB members worked together to identify and validate new promising drug targets and essential target-networks in various MPN. PL-D worked with candidate targets in a step-wise process: in a first step, candidate targets were examined for their expression in neoplastic cells in various mouse models, cell lines, and primary MPN cells, including LSC. The most promising targets and target-networks were also cross-validated, i.e. across the 4 MPN types, across various stages and phases of the disease, and across various cell types and cell lines. In a second step, candidate targets underwent validation using i) siRNA/shRNA and/or CRISPR/Cas9 and ii) various targeted drugs. Project parts #02, #04, and #10 worked together to examine knock-down effects in cell lines including LSC-like cell lines, #04, #10, and #11 worked together to apply shRNA and CRISPR/Cas9 in primary LSC, and #04, #06, #07, and #11 worked together to test and to validate the identified targets in various mouse models. Finally, critical target networks through which synergistic drug effects can be elicited were identified. The most effective drug combinations were then tested on primary neoplastic MPN cells, including drug-resistant sub-clones and LSC as well as normal (healthy) SC. Eventually, the most effective drug combinations were tested in suitable xenotransplantation models employing primary MPN cells, primary LSC, and/or various LSC-like cell lines. A complementary, patient-specific, screen approach, pharmacoscopy, was established by members of #11 in our SFB. In this assay, MPN cells and phenotypically defined LSC (but also normal SC) can be tested for their response against several hundred anti-neoplastic drugs in parallel. In addition, this high-capacity screen can test various drug combinations and can employ different read outs (cell survival, apoptosis, growth, cell-cell interactions) in a patient-specific manner. In the second funding period, this approach was extended to various MPN models, to primary LSC, and to the pre-testing of various drug combinations that was then applied to patients with advanced MPN. Drugs and drug combinations were selected for pharmacoscopy-testing based on the disease-model analyzed, literature data, and drugs developed in pre-clinical validation in various project parts in our SFB.