Basic Biology I
Despite significant advances in the treatment of leukemia over the past four decades, the rate of long-term survival has reached a plateau and still large numbers of leukemia patients die, mostly because of relapse and drug-resistance, which was recently attributed to the persistence of leukemia stem cells. If a therapy succeeds in eradicating leukemia stem cells, de novo initiation of the disease (relapse) is no longer possible. Therapeutic progress in recent clinical trials has likely been stalled, partly because current cytotoxic therapy approaches target proliferating bulk leukemia cells rather than quiescent leukemia stem cells. We now discovered that BCL6, a factor known to play a central role in B cell lymphomas, also plays a key role in the maintenance of leukemia stem cells. Since leukemia stem cells represent the origin of relapse and drug-resistance in leukemia in many cases, the identification of BCL6 as a target for leukemia stem cell eradication holds great promise. BCL6 is a master regulatory factor that controls the production of many different important genes. BCL6 was not previously known to be involved in leukemias. In preliminary studies for this proposal, we have discovered aberrant expression of BCL6 as a central component of a fundamentally novel pathway of leukemia stem cell self-renewal and drug-resistance in a wide array of human leukemias, some of which are still difficult to treat. In these leukemias, drug-treatment results in aberrant production of BCL6 by the leukemia cells, which appears to allow leukemia stem cell to self-renew and become resistance against drugtreatment. Recently a drug has been developed that can attach to BCL6 and block its cancer-causing activities. We found that this BCL6 inhibitor, which is called RI-BPI, has strong synergistic activity when combined with conventional drug-treatment, which opens up a powerful new therapeutic strategy for leukemia stem cell eradication through targeted inhibition of BCL6. Based on the discovery of BCL6 as a key component of a novel pathway of drug-resistance and stem cell self-renewal in a wide array of leukemias, we propose three Aims to develop these findings towards application in patient care: (1) To test the hypothesis that aberrant expression of BCL6 in human leukemia cells promotes leukemia stem cell survival, (2) To determine the frequency and phenotype of BCL6-dependent leukemia stem cells in human B cell ALL and (3) To validate a the role of the BCL6 inhibitor RI-BPI as a therapy for targeted eradication of leukemia stem cells. Since RI-BPI is currently going through the process of approval for use in clinical trials, we expect to be able to test the power of this approach in clinical trials by the end of the funding period.
Statement of Benefit to California:
B cell lineage acute lymphoblastic leukemia (ALL) represents the most frequent malignancy in childhood and is frequent in adults as well. Thousands of children and adults in California are afflicted with B cell leukemia and a significant portion of these patients will ultimately die despite the tremendous progress that has been made in leukemia treatment. Compelling evidence indicates that many leukemias are not curable because currently available chemotherapy target the bulk of the rapidly dividing leukemia cells but not the rare drug resistant leukemia stem cells that are quiescent and do not divide. For this reason, current research efforts both by laboratory investigators and clinicians focus on the leukemia stem cells because they are widely considered as the origin of drug-resistance and recurrence of the disease. Ground-breaking research in other subtypes of leukemia that are more frequent in adults has recently identified the leukemia stem cell. Additional research even found the "Achilles heel" of these resilient leukemia cells and potential targets for future drug-therapy. Unfortunately, leukemia stem cells have not been identified in B cell lineage ALL, nor do we know the mechanism that enables drug-resistance in these leukemia stem cells. The absence of this information represents a major unsolved problem, because knowledge about the biology of the leukemia stem cells is required for the development of future drug-therapies that will help to eradicate leukemia stem cells in this frequent leukemia subset. This seems particularly important, since B cell lineage ALL accounts for about 30% of all childhood cancers and is by far the most frequent malignancy in children and teenagers. In summary, the benefits to the citizens of California from the CIRM disease specific grant in leukemia are: (1) direct benefit to the thousands of patients with B cell lineage leukemia (2) higher quality of life due to definitive and targeted treatments that avoid severe side-effects and long-term disabilities (3) new partnership between the laboratory and clinical investigators as well as the pharmaceutical sector in California that leads to new synergies (4) the deployment of a multidisciplinary approach for pathwayspecific drug-therapy that will be applicable to other types of cancer (5) Realization of the CIRM mandate to develop highly effective novel therapies within a short time to benefit the health of Californians suffering from B cell lineage ALL.
The goal of this proposal is to examine the role of BCL6, a transcriptional repressor, in B cell lineage acute lymphoblastic leukemia (ALL) stem cell self-renewal and to validate BCL6 as a target for pathway-specific leukemia stem cell therapy. The frequency and the role of leukemic stem cells in propagating ALL and in conferring therapy resistance and recurrence are poorly understood. The central hypothesis is that chemotherapeutic tyrosine kinase inhibitors increase expression of BCL6 in B cell leukemias, activating an embryonic gene expression program resulting in leukemic stem cell survival and self-renewal which may lead to therapy resistance. In Aim 1, the applicant proposes to test this hypothesis by performing gain- and loss-of-function studies for BCL6 in primary human B cell lineage ALL cells using self-renewal assays and serial transplantation in immunodeficient mice as readouts. In Aim 2, the applicant will use a novel BCL6 reporter system to determine the frequency of BCL6-dependent leukemic stem cells in patient-derived B cell lineage ALL and establish surrogate makers suitable for flow cytometry. Finally, in Aim 3, the applicant proposes to validate BCL6 as a target for therapy in xenografted immunodeficient mouse models. Reviewers agreed that this proposal addresses a major unsolved problem of ALL drug resistance and could have a significant impact. ALL is the most common childhood malignancy and there is a need to develop more efficacious therapies to treat this disease. Reviewers found the proposal to be quite innovative, as it focuses on a fundamentally novel signaling pathway in leukemia stem cells and employs a creative approach. One reviewer noted that the proposal is very focused on basic mechanisms of ALL generation, maintenance and drug resistance. Another reviewer questioned whether ALL adheres to the cancer stem cell model and thus whether this is really a stem cell proposal but other reviewers argued affirmatively on both points. Reviewers expressed a number of concerns about the research plan that dampened their enthusiasm for the proposal. Overall, they found it difficult to read and extraordinarily dense but lacking sufficient background information and experimental detail. One reviewer would have appreciated a clear description of the viral vector at the beginning of Aim 1, the gradual disclosure of relevant features made evaluation of this aim difficult. This reviewer also noted that the readout of leukemic stem cell frequency in patient samples in Aim 1 is not very quantitative. The reviewer suggested that the proposed serial transplantation experiments should include limiting cell dilution transplants to provide a more quantitative measure of leukemic stem cell frequency as well as their capacity for sustained self-renewal. Also in Aim 1, the rationale for studying 25 primary human ALL samples is not clearly stated. If fewer samples could be reliably used, this would lessen the time and cost of the proposed research. With regard to Aim 2, one reviewer praised the creative approach but another felt that the rationale for pursuing this specific aim was not well justified. The reviewers found Aim 3 to be extraordinarily ambitious, involving identification and characterization of the biological effect of BCL6 target gene expression in ALL patient samples. One reviewer felt that it lacked sufficient detail with regard to how many human samples will be analyzed, as well as the duration and concentrations of the inhibitors to be used. Moreover, verification of the identified target genes is not explicitly proposed. A reviewer also noted that the functional characterization proposed in Aim 3 appears to be limited to experiments that would prove the applicant’s hypothesis, potentially biasing interpretation. Reviewers did note that this proposal is supported by a wealth of preliminary data and the rationale for the proposed experiments is generally sound. Reviewers agreed that the applicant is well qualified to conduct the proposed research. They particularly appreciated the high percent effort the PI proposes to devote to the project. Reviewers praised the assembled research team and appreciated the letters of collaboration, although many of those letters express support for a grant submitted to another funding agency. Overall, while reviewers appreciated that this proposal describes an innovative approach to study a major unsolved problem, they raised significant doubts about its feasibility. Reviewers described the research plan as poorly organized and extraordinarily ambitious, and noted that certain proposed experiments lacked sufficient detail.