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 will further develop this concept in the second SFB period by studying genetic, epigenetic, and somatic lesions in an integrative approach that takes multiple factors and features as well as sub-clone formation in MPN into account. The two CF-PF of the SFB will again support these efforts and will cooperate closely with #02, #04, and #10 in this PL. Germline variations and gender-related aspects of MPN will be studied in project #02, #04, and #09, and new somatic lesions and epigenetic patterns and targets in #02, #04, and #10. Sequencing studies will focus on genes involved in the regulation of growth, survival, and distribution of LSC in MPN. In each case, sequencing results will be correlated with protein expression profiles, data obtained in functional assays, and clinical features and endpoints including survival, progression, and drug responses. The 2 CF-PF will closely collaborate with individual project parts and clinical partners in these analyses. In #02 and #04, SFB members will also try to identify early stages of MPN evolution where LSC-derived sub-clones remain small-sized for some time before they expand to an overt neoplasm. Here, the major aim will be to define epigenetic and somatic events contributing to sub-clone-expansion and disease-manifestation.
PL-B. Functional analysis of MPN development and progression: cellular hierarchies and MPN sub-clone formation, LSC-niche interactions, and the cytokine network.
As in the first funding period, PL-B will address the cellular complexity in various MPN, and will continue 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 will closely collaborate 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 will screen for novel specific SC markers in the second funding period. As in the previous SFB phase, PL-B will study 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 will study cytokines and cytokine-networks suppressing immune responses by upregulating certain recognition-receptors, like CD47, or immune checkpoint receptors, like PD-L1 (CD274) on LSC. For example, it remains unknown whether IFN-gamma signaling regulates CD274 expression in MPN LSC in the same way as in AML cell lines. PL-B will also explore the cellular source of cytokines, with special emphasis on neoplastic cells. For example, basophils typically expand in advanced CML and are a rich source of angiogenic cytokines, like VEGF or HGF67; and niche cell-derived OSM has been identified as an oncogene-dependent trigger of angiogenesis and fibrosis in JAK2- and PDGFRA-mutated MPN in our consortium. In the second period of F47, these studies will continue and cytokine expression levels will be correlated with disease-pathology (fibrosis, angiogenesis), specific functions of LSC, and with growth and survival of niche cells. Finally, PL-B will study LSC sub-clone formation and the trans-differentiation capacity of LSC (#04). Sub-clone-evolution and expansion will be 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 will be 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 will be 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.
As in the first funding period, PL-C will focus 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 should be identified in MPN (stem) cells in #02 through #11 in collaboration with the biobank and omics CF-PF of our SFB. Depending on the project part and disease-model, several different strategies and approaches will be employed to identify critical signaling nodes and key effector molecules. In several instances, relevant molecules will first be identified in murine MPN models (#06, #07, #10). In this case, expression and function of newly identified molecules or pathways will be confirmed in human cell lines and primary MPN cells (LSC) or niche cells in SFB collaborations. In other projects, signaling molecules or effector molecules will be identified in human MPN cells and will then be validated in appropriate mouse models. Expression and function of these molecules will be 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 will be explored using inhibitory antibodies, shRNA or CRISPR/Cas9, or drugs directed against ligand-proteins or receptors. The impact of various signaling molecules will be determined by using shRNA, CRISPR/Cas9, as well as specific chemical compounds. One critical aim in PL-C is 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 will be applied to address this objective in the second SFB period. This will be 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 will continue to work together to identify new promising drug targets and essential target-networks in various MPN and will validate those targets and pathways that have been identified in neoplastic (stem) cells in the first SFB period (see individual project parts). The master plan of the SFB will provide a solid basis for these studies. The operational plan for PL-D will again be to work with candidate targets in a step-wise process: in a first step, candidate targets will be 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 will also be 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 will undergo validation using i) siRNA/shRNA and/or CRISPR/Cas9 and ii) various targeted drugs. Project parts #02, #04,and #10 will work together to examine knock-down effects in cell lines including LSC-like cell lines, #04, #10, and #11 will work together to apply shRNA and CRISPR/Cas9 in primary LSC, and #04, #06, #07, and #11 will work 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 should be identified. The most effective drug combinations will then be tested on primary neoplastic MPN cells, including drug-resistant sub-clones and LSC as well as normal (healthy) SC. In a final step, the most effective drug combinations will be 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, has been 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.65 In the second funding period, this approach will be extended to various MPN models, to primary LSC, and to the pre-testing of various drug combinations that will later be applied to patients with advanced MPN. Drugs and drug combinations will be 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.