Periodic Reporting for period 4 - LeukaemiaTargeted (Selecting genetic lesions with essential function for patients' leukaemia in vivo as targets for precision medicine)
Período documentado: 2021-02-01 hasta 2021-07-31
In Europe, around two million individuals die from cancer each year. Cancer is a genetic disease and each patient's tumour contains several genetic lesions which are identified by next generation sequencing (NGS) and influence patient's outcome. A global current challenge lies in translating NGS data into benefit of cancer patients.
As attractive novel therapeutic concept, precision medicine addresses genetic lesions using targeted therapies. A large number of targeted drugs and compounds exist and are currently developed such as kinase inhibitors; unfortunately, numerous clinical trials on targeted therapies failed.
In order to better exploit NGS data, it is important to discriminate between genetic lesions that are required and maintain patients' tumours in vivo and others that do not – an impossible mission so far. My proposal aims at solving this key question.
Using acute leukaemia as model tumour disease, we propagate primary tumour cells from patients in immuno-deficient mice. We recently pioneered a worldwide unique technique which allows the distinct genetic manipulation of individual patients' tumour cells while they grow in vivo.
We will molecularly target tumour-specific genetic lesions one by one; if tumour load is reduced, the lesion fulfils an essential function; essential lesions represent attractive therapeutic targets. Using our cutting edge technology, we will identify genetic lesions with essential, tumour-relevant function
(i) in established tumour disease and
(ii) in the clinically challenging situations of minimal residual disease and relapse.
Our approach implements a new paradigm for target selection in oncology. Our work introduces molecular target validation as important step into the value chain of precision medicine which will tailor drug development by industry and academia. Our approach will improve patient care and the success rate of clinical trials for the benefit of patients suffering acute leukaemia and putatively other cancers.
As attractive novel therapeutic concept, precision medicine addresses genetic lesions using targeted therapies. A large number of targeted drugs and compounds exist and are currently developed such as kinase inhibitors; unfortunately, numerous clinical trials on targeted therapies failed.
In order to better exploit NGS data, it is important to discriminate between genetic lesions that are required and maintain patients' tumours in vivo and others that do not – an impossible mission so far. My proposal aims at solving this key question.
Using acute leukaemia as model tumour disease, we propagate primary tumour cells from patients in immuno-deficient mice. We recently pioneered a worldwide unique technique which allows the distinct genetic manipulation of individual patients' tumour cells while they grow in vivo.
We will molecularly target tumour-specific genetic lesions one by one; if tumour load is reduced, the lesion fulfils an essential function; essential lesions represent attractive therapeutic targets. Using our cutting edge technology, we will identify genetic lesions with essential, tumour-relevant function
(i) in established tumour disease and
(ii) in the clinically challenging situations of minimal residual disease and relapse.
Our approach implements a new paradigm for target selection in oncology. Our work introduces molecular target validation as important step into the value chain of precision medicine which will tailor drug development by industry and academia. Our approach will improve patient care and the success rate of clinical trials for the benefit of patients suffering acute leukaemia and putatively other cancers.
In the first 30 months of funding, we have established primary tumor cells from numerous patients with acute leukaemias (AL) on immune-incompetent mice to generate patient-derived xenograft (PDX) models. In parallel, we have described all genetic lesions of each AL sample using next generation sequencing. We have used genetic engineering using lentiviruses and have molecularly modified established PDX cells. We have described two novel, yet unknown vulnerabilities of PDX AL models, namely the anti-apoptotic protein XIAP as well as the transcription factor KLF4 (manuscripts in preparation). Our data suggest using Smac mimetics targeting XIAP in patients with overexpressed XIAP as well as Azacytidin in acute lymphoblastic leukemia with downregulated KLF4.
Acute leukemias represent the single most frequent malignant disease in children and suffer poor prognosis, especially upon disease relapse and in adult patients. The aim of the ERC CoG grant LeukemiaTargeted was to improve anti-cancer treatment by identifying and characterizing lesions with essential, tumour-relevant function in patients-tumor cells in vivo, focussing on treatment resistant and dormant leukemia cells, as well as leukemia stem cells. The aim is to use such vulnerabilities as therapeutic targets for developing innovative targeted anti-cancer therapies for translation into the clinics.
Since start of the ERC CoG grant and as technical prerequisite to our planned work, we advanced genetic engineering in patient-derived xenograft (PDX) cells to study molecularly modified PDX models in vivo.
1. We introduced inducible systems for Cre-ERT2-loxP mediated knockdown (Carlet et al., Jeremias, Nature Comm., 2021).
2. We established CRISPR/Cas9 mediated knockout and a reporter system for enriching PDX cells with successful knockout (Liu et al., Jeremias, Scientific Reports, 2020).
3. We established a technique for cloning of several customized libraries in parallel (Becker et al, Jeremias*, Braun*, Nucleic Acids Res., 2019; * shared last authorship).
4. We established CRISPR/Cas9 mediated dropout screens in PDX models in vivo (Bahrami et al., Jeremias, ASH abstract at Blood, 2019; manuscript in preparation).
5. We established multiplexed competitive in vivo approaches using up to 5 fluorochromes (Zeller et al., Jeremias, ASH abstract at Blood, 2019; manuscript in revision).
Complex genetic engineering in leukemia PDX cells and for in vivo use allowed insights into several known and novel vulnerabilities with essential function. We studied their essential function for spontaneous growth of PDX leukemia in vivo and in resistant and/or dormant and/or leukemia stem cells. While all mentioned vulnerabilities represent bona fide therapeutic targets in defined clinical situations, our approaches allowed identifying those which are most attractive and stronger than others. We discovered several unexpected biologic insights.
1. In PDX leukemia in vivo, dormancy represents a reversible, plastic phenotype, opening new therapeutic avenues (Ebinger et al., Jeremias, Cancer Cell 2016, IF 27,4; Ebinger et al., Jeremias, Haematologica, 2020, IF 7,1; Senft, Jeremias, Dev. Cell, 2019).
2. Molecular re-expression by inducible expression in PDX models or treatment with Azacytidine restore expression of downregulated KLF4 and leads to reduced leukemia growth, especially after the situation of minimal residual disease (Liu et al, Jeremias, Biomarker Res, 2020).
3. Loss of EZH2 induces resistance towards conventional chemotherapy in acute leukemias (Göllner et al., Jeremias et al., Müller-Tidow, Nature Medicine, 2017).
4. XIAP represents a novel vulnerability in acute leukemia and sensitizes towards conventional chemotherapy in vivo (Vergalli et al., Jeremias, abstract in Eur J Cancer, 2017; manuscript in revision).
5. In PDX cells in vivo, but not in cell line cells in vitro, WT1 and DNMT3A harbour an essential role for leukemic growth, including leukemia stem cells (Ghalandary et al. Jeremias; abstract selected for oral presentation at the annual ASH meeting 2021; manuscript in preparation).
6. Leukemia cell homing to the bone marrow depends on presence of ADAM10 which presents a novel vulnerability and therapeutic target (Bahrami et al., abstract selected for oral presentation at the annual ASH meeting 2021; manuscript in preparation).
7. In vivo acquired treatment resistance in PDX models is associated by heterogenous genetic alterations, but all subclones respond to knockout of BCL-2 or treatment with Venetoclax (Wirth et al., Jeremias; several oral presentations; manuscript in preparation).
In conclusion, the ERC CoG grant enabled us to identify novel vulnerabilities and to specify the role of known signalling molecules with essential function. Our results harbour the potential to lead into novel treatment approaches for acute leukemias and well beyond in other types of cancer.
Since start of the ERC CoG grant and as technical prerequisite to our planned work, we advanced genetic engineering in patient-derived xenograft (PDX) cells to study molecularly modified PDX models in vivo.
1. We introduced inducible systems for Cre-ERT2-loxP mediated knockdown (Carlet et al., Jeremias, Nature Comm., 2021).
2. We established CRISPR/Cas9 mediated knockout and a reporter system for enriching PDX cells with successful knockout (Liu et al., Jeremias, Scientific Reports, 2020).
3. We established a technique for cloning of several customized libraries in parallel (Becker et al, Jeremias*, Braun*, Nucleic Acids Res., 2019; * shared last authorship).
4. We established CRISPR/Cas9 mediated dropout screens in PDX models in vivo (Bahrami et al., Jeremias, ASH abstract at Blood, 2019; manuscript in preparation).
5. We established multiplexed competitive in vivo approaches using up to 5 fluorochromes (Zeller et al., Jeremias, ASH abstract at Blood, 2019; manuscript in revision).
Complex genetic engineering in leukemia PDX cells and for in vivo use allowed insights into several known and novel vulnerabilities with essential function. We studied their essential function for spontaneous growth of PDX leukemia in vivo and in resistant and/or dormant and/or leukemia stem cells. While all mentioned vulnerabilities represent bona fide therapeutic targets in defined clinical situations, our approaches allowed identifying those which are most attractive and stronger than others. We discovered several unexpected biologic insights.
1. In PDX leukemia in vivo, dormancy represents a reversible, plastic phenotype, opening new therapeutic avenues (Ebinger et al., Jeremias, Cancer Cell 2016, IF 27,4; Ebinger et al., Jeremias, Haematologica, 2020, IF 7,1; Senft, Jeremias, Dev. Cell, 2019).
2. Molecular re-expression by inducible expression in PDX models or treatment with Azacytidine restore expression of downregulated KLF4 and leads to reduced leukemia growth, especially after the situation of minimal residual disease (Liu et al, Jeremias, Biomarker Res, 2020).
3. Loss of EZH2 induces resistance towards conventional chemotherapy in acute leukemias (Göllner et al., Jeremias et al., Müller-Tidow, Nature Medicine, 2017).
4. XIAP represents a novel vulnerability in acute leukemia and sensitizes towards conventional chemotherapy in vivo (Vergalli et al., Jeremias, abstract in Eur J Cancer, 2017; manuscript in revision).
5. In PDX cells in vivo, but not in cell line cells in vitro, WT1 and DNMT3A harbour an essential role for leukemic growth, including leukemia stem cells (Ghalandary et al. Jeremias; abstract selected for oral presentation at the annual ASH meeting 2021; manuscript in preparation).
6. Leukemia cell homing to the bone marrow depends on presence of ADAM10 which presents a novel vulnerability and therapeutic target (Bahrami et al., abstract selected for oral presentation at the annual ASH meeting 2021; manuscript in preparation).
7. In vivo acquired treatment resistance in PDX models is associated by heterogenous genetic alterations, but all subclones respond to knockout of BCL-2 or treatment with Venetoclax (Wirth et al., Jeremias; several oral presentations; manuscript in preparation).
In conclusion, the ERC CoG grant enabled us to identify novel vulnerabilities and to specify the role of known signalling molecules with essential function. Our results harbour the potential to lead into novel treatment approaches for acute leukemias and well beyond in other types of cancer.