Mechanism of BCL6-dependent stem cell maintenance in B cell lineage leukemia

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01358
ICOC Funds Committed: 
$0
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
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.
Progress Report: 
  • Parkinson's disease results primarily from the loss of neurons deep in the middle part of the brain (the midbrain), in particular neurons that produce dopamine (referred to as “dopaminergic”). In this region of the midbrain there are actually two different groups of dopaminergic (DA) neurons, and only one of them, the neurons of the substantia nigra (SN) are highly susceptible to degeneration in patients with PD. There is a relative sparing of the second group of midbrain dopaminergic neurons, called the ventral tegmental area (VTA) dopaminergic neurons. These two groups of neurons reside close to each other in the brain and both make dopamine. They are virtually indistinguishable except for one major functional difference—they release dopamine, the transmitter that is lost in Parkinson’s patients, to their downstream neuronal targets in different ways. SN neurons deliver dopamine in small rapid squirts, like a sprinkler, whereas VTA neurons have a tap that provides a continuous stream of dopamine.
  • A major therapeutic strategy for patients with PD is to make new DA neurons from human embryonic stem cells (hES). As stem cells have the potential to develop into any type of cell in the body, these considerations suggest that we should devise a way to produce SN neurons in the absence of VTA neurons from stem cells for use in transplantation. At present although we can produce dopaminergic neurons from hES cells, the scientific community cannot distinguish SN from VTA neurons in vitro due to lack of molecular markers or a bioassay, and we are therefore unable to identify culture conditions that favor the production of one over the other,
  • In addition to releasing dopamine differently, SN and VTA neurons have axons that project to different regions of the striatum. It has been shown over the last decade that specific classes of guidance cues guide axons to their particular targets. One approach we have taken has been to investigate whether differences in axon guidance receptor expression and or responses to guidance cues in vitro might provide both markers and a bioassay that will distinguish SN from VTA neurons. Over the last year we have shown that VTA and SN neurons respond differentially to Netrin-1 and express different markers associated with the guidance cue family. We now have a bioassay and markers that distinguish these two populations of neurons in vitro and in the coming year we plan to utilize this information to identify cultures conditions that favor the production of SN over VTA neurons, from hES cells.
  • Parkinson’s disease results primarily from the loss of neurons deep in the middle part of the brain (the midbrain), in particular neurons that produce dopamine (referred to as “dopaminergic”). In this region of the midbrain there are actually two different groups of dopaminergic (DA) neurons, and only one of them, the neurons of the substantia nigra (SN) are highly susceptible to degeneration in patients with PD. There is a relative sparing of the second group of midbrain dopaminergic neurons, called the ventral tegmental area (VTA) dopaminergic neurons. These two groups of neurons reside close to each other in the brain and both make dopamine. They are virtually indistinguishable except for one major functional difference—they release dopamine, the transmitter that is lost in Parkinson’s patients, to their downstream neuronal targets in different ways. SN neurons deliver dopamine in small rapid squirts, like a sprinkler, whereas VTA neurons have a tap that provides a continuous stream of dopamine. 
A major therapeutic strategy for patients with PD is to make new DA neurons from human embryonic stem cells (hES). As stem cells have the potential to develop into any type of cell in the body, these considerations suggest that we should devise a way to produce SN neurons in the absence of VTA neurons from stem cells for use in transplantation. At present although we can produce dopaminergic neurons from hES cells, the scientific community cannot distinguish SN from VTA neurons in vitro due to lack of molecular markers or a bioassay, and we are therefore unable to identify culture conditions that favor the production of one over the other, 
In addition to releasing dopamine differently, SN and VTA neurons have axons that project to different regions of the striatum. It has been shown over the last decade that specific classes of guidance cues guide axons to their particular targets. One approach we have taken has been to investigate whether differences in axon guidance receptor expression and or responses to guidance cues in vitro might provide both markers and a bioassay that will distinguish SN from VTA neurons. We showed previously that VTA and SN neurons respond differentially to Netrin-1 and express different markers associated with the guidance cue family. Also, in this year using backlabeling, laser capture and microarray analysis of SN vs VTA neurons, we have identified a number of genes expressed in on or the other population. We now have a bioassay and markers that distinguish these two populations of neurons in vitro and in the coming year we plan to utilize this information to identify cultures conditions that favor the production of SN over VTA neurons, from hES cells.
  • Parkinson's disease (PD) is a neurodegenerative movement disorder that affects more than six million people worldwide. The main symptoms of the disease result from the loss of neurons from the midbrain that produce dopamine (referred to as "dopaminergic" or DA neurons).Human embryonic stem cells (hESC) offer an exciting opportunity to treat Parkinson’s disease by transplanting hESC-derived DA neurons to replace those that have died. There are actually two groups of midbrain DA neurons in the human brain. Those from the substantia nigra (SN) are highly susceptible to degeneration in Parkinson's patients while those from the ventral tegmental area (VTA) are not. These two types of neurons have similar features but have different functions and it is important to ensure that DA neurons from hESC are the correct SN type before they are used in therapy. The primary goal of this research was to study these two neuronal types in animals and determine if the distinguishing features discovered in mice or rats can be used to more easily recognize and purify SN-type DA neurons made from hESC.
  • One of the discoveries made in this research is that SN and VTA neurons show differences in how they make connections within the brain. We have been able to identify some of the molecules that guide each neuron to connect to it appropriate target and have found that SN and VTA neurons placed in the petri dish can be distinguished from each other by their response to guidance molecules. Work in the final period of this grant has focused on testing guidance response in hESC-derived DA neurons and we have found that many of the neurons produced from hESC do show SN-like responses to guidance molecules. This discovery is being further developed as a screening tool to help guide our ongoing efforts to make increasingly pure populations of DA neurons from hESC.
  • Future human trials will likely utilize such DA neurons but since embryonic stem cells have the potential to develop into any type of cell in the body, it is important to ensure that the production methods used to make a therapeutic product for Parkinson’s disease do indeed specifically produce SN neurons. Prior to the research supported under this CIRM grant, the scientific community was not able to distinguish SN from VTA neurons outside of their normal brain environment and therefore had no ability to confirm whether a method produced one type selectively and not the other. Further refinements of the assay tools developed in our research may provide a practical means of quantifying the purity of a DA neuron preparation. This would have a significant impact transplantation therapy as well as provide useful insights into the molecular mechanisms that underlie proper connectivity and function of SN and VTA DA neurons in humans.

© 2013 California Institute for Regenerative Medicine