Dual targeting of tyrosine kinase and BCL6 signaling for leukemia stem cell eradication

Dual targeting of tyrosine kinase and BCL6 signaling for leukemia stem cell eradication

Funding Type: 
Early Translational II
Grant Number: 
TR2-01816-B
Award Value: 
$2,756,536
Disease Focus: 
Blood Cancer
Cancer
Collaborative Funder: 
Germany
Stem Cell Use: 
Cancer Stem Cell
Cell Line Generation: 
Adult Stem Cell
Cancer Stem Cell
Status: 
Active
Public Abstract: 
Leukemia is the most frequent form of cancer in children and teenagers, but is also common in adults. Chemotherapy has vastly improved the outcome of leukemia over the past four decades. However, many patients still die because of recurrence of the disease and development of drug-resistance in leukemia cells. In preliminary studies for this proposal we discovered that in most if not all leukemia subtypes, the malignant cells can switch between an “proliferation phase” and a “quiescence phase”. The “proliferation phase” is often driven by oncogenic tyrosine kinases (e. g. FLT3, JAK2, PDGFR, BCR-ABL1, SRC kinases) and is characterized by vigorous proliferation of leukemia cells. In this phase, leukemia cells not only rapidly divide, they are also highly susceptible to undergo programmed cell death and to age prematurely. In contrast, leukemia cells in “quiescence phase” divide only rarely. At the same time, however, leukemia cells in "quiescence phase" are highly drug-resistant. These cells are also called 'leukemia stem cells' because they exhibit a high degree of self-renewal capacity and hence, the ability to initiate leukemia. We discovered that the BCL6 factor is required to maintain leukemia stem cells in this well-protected safe haven. Our findings demonstrate that the "quiescence phase" is strictly dependent on BCL6, which allows them to evade cell death during chemotherapy treatment. Once chemotherapy treatment has ceased, persisting leukemia stem cells give rise to leukemia clones that reenter "proliferation phase" and hence initiate recurrence of the disease. Pharmacological inhibition of BCL6 using inhibitory peptides or blocking molecules leads to selective loss of leukemia stem cells, which can no longer persist in a "quiescence phase". In this proposal, we test a novel therapeutic concept eradicate leukemia stem cells: We propose that dual targeting of oncogenic tyrosine kinases (“proliferation”) and BCL6 (“quiescence”) represents a powerful strategy to eradicate drug-resistant leukemia stem cells and prevent the acquisition of drug-resistance and recurrence of the disease. Targeting of BCL6-dependent leukemia stem cells may reduce the risk of leukemia relapse and may limit the duration of tyrosine kinase inhibitor treatment in some leukemias, which is currently life-long.
Statement of Benefit to California: 
Leukemia represents the most frequent malignancy in children and teenagers and is common in adults as well. Over the past four decades, the development of therapeutic options has greatly improved the prognosis of patients with leukemia reaching 5 year disease-free survival rates of ~70% for children and ~45% for adults. Despite its relatively favorable overall prognosis, leukemia remains one of the leading causes of person-years of life lost in the US (362,000 years in 2006; National Center of Health Statistics), which is attributed to the high incidence of leukemia in children. In 2008, the California Cancer Registry expected 3,655 patients with newly diagnosed leukemia and at total of 2,185 death resulting from fatal leukemia. In addition, ~23,300 Californians lived with leukemia in 2008, which highlights that leukemia remains a frequent and life-threatening disease in the State of California despite substantial clinical progress. Here we propose the development of a fundamentally novel treatment approach for leukemia that is directed at leukemia stem cells. While current treatment approaches effectively diminish the bulk of proliferating leukemia cells, they fail to eradicate the rare leukemia stem cells, which give rise to drug-resistance and recurrence of the disease. We propose a dual targeting approach which combines targeted therapy of the leukemia-causing oncogene and the newly discovered leukemia stem cell survival factor BCL6. The power of this new therapy approach will be tested in clinical trials to be started in the State of California.
Progress Report: 

Year 1

During the past reporting period (months 18-24 of this grant), we have made progress towards all three milestones. Major progress in Milestone 1 was made by identifying 391 compounds in 10 lead classes that will be developed further in a secondary fragment-based screen. While the goal of identifying lead class compounds with BCL6 inhibitory activity has already been met, we propose to run a secondary, fragment-based screen to refine the existing lead compounds and prioritize a small number for cell-based validation in Milestone 2. The success in Milestone 1 was based on computational modeling, HTS of 200,000 compounds and Fragment-based drug discovery (FBDD). For Milestone 2, we have successfully established POC analysis tools for validation of the ability of compounds to bind the BCL6 lateral groove and already produced 300 mg of BCL6-BTB domain protein needed for biochemical binding assays. Progress in Milestone 2 is based on surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) assays. In the coming months, we will use crystallographic fragment screening using a subset of our fragment library in addition to SPR and NMR, since crystallographic fragment screens have been shown to yield complimentary hits. For Milestone 3, we have now set up a reliable method to measure disease-modifying activity of BCL6-inhibitory compounds based on a newly generated knockin BCL6 reporter mouse model, in which transcriptional activation of the endogenous BCL6 promoter drives expression of mCherry. This addresses a main caveat of these measurements was that they were strongly influenced by the copy number of lentivector integrations. The BCL6fl/+-mCherry knockin BCL6 reporter system will provide a stable platform to study BCL6-expressing leukemia cells and effects of BCL6 small molecule inhibitors on survival and proliferation on BCL6-dependent leukemia cell populations. This will be a key requirement to measure disease-modifying activity of inhibitory compounds in large-scale assays in Milestone 3. Other requirements (e.g. leukemia xenografts) are already in place. 

© 2013 California Institute for Regenerative Medicine