Overview: Dr. Perl's request, entitled: "Validating mTOR as a therapeutic target in AML"
Explained as follows: Our laboratory and clinical group have had a longstanding interest in mechanisms through which leukemia cells reprogram their response to environmental triggers in ways that promote their cell growth and survival. By inhibiting such biologic processes with novel drugs, we seek to enhance anti-leukemic effects with less toxicity than standard chemotherapy. We previously demonstrated that acute myeloid leukemia (AML) cells show inappropriate activation of a series of interacting enzymes called the PI3Kinase/AKT/mTOR pathway. By activating this pathway, AML cells maintain a growth and survival response despite cellular stresses and toxins such as chemotherapy. Although mTOR inhibitors alone have modest anti-leukemic activity, we established that treating AML cells with mTOR inhibitors such as rapamycin (sirolimus) dramatically enhances their sensitivity to cytotoxic chemotherapy. We then performed clinical trials combining sirolimus with intensive chemotherapy in patients with high-risk AML. These studies showed our approach was feasible, tolerable, and clinically active. During these trials, we optimized measurement of the mTOR enzyme in leukemia cells by a novel application of flow cytometry called phospho-flow. With this approach, we demonstrated that activation of mTOR was variable across patients, with some showing a marked increase of mTOR enzyme activity and others showing little to no activation. Importantly, approximately 40% of patients' leukemia cells showed mTOR activation prior to therapy and obvious inhibition during sirolimus therapy. For this sub-group of patients, the response rate was approximately double that of patients who lack mTOR activation or who showed resistance to the targeted drug. This observation provides clinical support for our initial hypothesis that mTOR inhibition enhances chemotherapy response. As the flow cytometric analysis is performed in real time and is evaluable within 3 days, we hypothesize that our assay may serve as a screening test to allocate patients to personalized approaches. The project involves ongoing clinical trial development of mTOR inhibitors in AML along with flow cytometric bioassays to identify patients most likely to respond to this novel treatment approach.
The Problem: Myelodysplastic Syndrome (MDS) is an extremely variable pre-cancerous disorder of the blood. As this disorder progresses, about one third of the MDS patients develop a life-threatening cancer called acute myeloid leukemia (AML), for which there are few treatment options. MDS is thought to occur because the stem cells that form all blood, termed hematopoietic stem cells (HSC), are defective. Mutations in ribosomal proteins (RPs) cause diseases collectively termed ribosomopathies, which are associated with increased risk for development of MDS. For example, RPS14 haploinsufficiency has been shown to be responsible for bone marrow failure and increased cancer risk in the 5q- subset of Myelodysplastic Syndrome (MDS) patients. However, the precise mechanism by which inactivation of RPs contributes to the development of MDS-hematopoietic stem cells (HSC) and progression to AML has remained unclear. Our goal is that new treatment options could be discovered if the processes that cause MDS and AML were better understood. Our work supported by When Everyone Survives seeks to understand how the antagonistic functions of novel ribosomal protein paralogs Rpl22 and Rpl22-like1 (Like1) regulate hematopoietic stem cell function and transformation.
The Result: We have found that one cause of the defective function of HSC in MDS is inactivation of proteins that make up a cellular machine called the ribosome. Using the zebrafish and mouse model systems, we have found that two such ribosomal proteins (RP), called Rpl22 and Rpl22- like1 (Like1), must be carefully balanced in order to produce HSC and normal blood components. With the support from WES, we sought to determine how Rpl22 and Like1 contribute to MDS pathogenesis. And now we have made the surprising and novel observation that Rpl22 and Like1 also regulate pre-mRNA splicing.
What did we learn? Our findings revealed that imbalances of Rpl22 and Like1 can cause mis-splicing of essential transcriptional and epigenetic factors implicated in the pathogenesis of MDS/AML. We also elucidated the molecular basis by which the highly homologous ribosomal proteins, Rpl22 and Like1, and were able to perform antagonistic functions and assess how their regulation of premRNA splicing impacts hematopoiesis and contributes to the pathogenesis of MDS and AML.
Where do we go from here? Based on our results, which gained insight into the molecular pathways controlled by Rpl22 and Rpl22-like1, we expect that our analysis of the genetic and epigenetic pathways controlled by Rpl22 and Like1 in RNA splicing will reveal novel biomarkers for human MDS/AML, as well as novel targets against which therapies can be developed to more effectively treat MDS/AML patients.
The Problem: The goal of this project was to conduct preclinical testing of a novel combination of anti-leukemia drugs in a high-risk subset of pediatric leukemia. The most common cancer in children is B-cell acute lymphoblastic leukemia (B-ALL). Although current treatments cure most patients, around 10-15% relapse and have a poor prognosis. Recent cancer genomic studies have identified a subgroup of these high-risk B-ALL cases that are known as “Ph-like B-ALL”. An important molecular feature of these leukemias is the activation of a class of enzymes known as tyrosine kinases. Consequently, there is hope that tyrosine kinase inhibitors (TKIs) can provide effective treatments for Ph-like B-ALL patients. Another key feature of Ph-like B-ALL is high activity of an enzyme known as mTOR, which can be inhibited by drugs known as TOR-KIs. We propose that combining TKIs and TOR-KIs will be more effective than either drug alone.
Much of our efforts were directed to establishing experimental approaches. We were able to confirm that leukemia cells from children with Ph-like B-ALL can divide and increase in number after injection into mice. This is important because it provides a means to continue studying these rare cells that do not grow in vitro. A second advance was that we could measure the activity of tyrosine kinases and mTOR inside the leukemia cells and quantify changes that occur after treatment with the anti-cancer agents. The data support our hypothesis that combined treatment with TKIs and TOR-KIs suppresses activation of key cellular components more completely than either drug alone.
What We Learned: We obtained preliminary evidence to support our hypothesis that combining TKIs with TOR-KIs has a greater impact on leukemia cells than either drug alone. We also established experimental conditions that will allow us to conduct more extensive studies. Overall, we obtained encouraging evidence that we will be able to validate this drug combination as a promising therapy for Ph-like B-ALL.
Where do we go from here: The Principal Investigator has obtained additional funding for this project and will continue to address the objectives of the study.