Himalee Sabnis

Two mutations in the KINDLIN3 gene of a new leukocyte adhesion deficiency-III patient reveal distinct effects on leukocyte function in vitro

In the disorder leukocyte adhesion deficiency III (LAD-III), integrins on platelets and leukocytes are expressed but fail to function and this leads to severe bleeding and infections at an early age. Mutation in the KINDLIN3 (FERMT3) gene is the cause of LAD-III in patients from the Middle East, Malta, and Turkey. We describe 2 novel homozygous mutations in the KINDLIN3 gene of a new African-American patient that destabilize KINDLIN3 mRNA leading to loss of kindlin-3 protein. Transfection of wild-type (WT) KINDLIN3 cDNA restored integrin-related adhesion and migration in the LAD-III patients T and B lymphocytes. We analyzed the individual mutations separately in vitro to learn more about the function of the kindlin-3 protein. The first G>A mutation gives rise to a Gly308Arg change at the end of FERM (protein 4.1, ezrin, radixin, moesin) subdomain 2, and the second mutation is a base deletion causing early termination within the pleckstrin homology (PH) domain. This second mutation prevented membrane association of kindlin-3 and did not restore either adhesion or migration, whereas the FERM subdomain 2 mutation affected only migration. Thus, these LAD-III patient mutations have highlighted functionally important regions of kindlin-3 that alter leukocyte integrin-dependent function in 2 distinct ways.

Leukocyte adhesion deficiency‐III in an African‐American patient

Leukocyte adhesion deficiency‐III (LAD‐III) is a rare disorder characterized by abnormal signaling to β integrins, leading to defective leukocyte adhesion and chemotaxis and platelet aggregation. Here we present the first case of an African‐American female infant with this disorder. She had history of multiple infections, bleeding, and leukocytosis since birth. She was successfully treated with allogeneic bone marrow transplant using a reduced intensity‐conditioning regimen. Mutations in KINDLIN‐3 have been described in LAD‐III but the mutations in KINDLIN‐3 in her case are unique. Pediatr Blood Cancer 2010;55:180–182.

Molecular Targets for the Treatment of Juvenile Myelomonocytic Leukemia

Significant advances in our understanding of the genetic defects and the pathogenesis of juvenile myelomonocytic leukemia (JMML) have been achieved in the last several years. The information gathered tremendously helps us in designing molecular targeted therapies for this otherwise fatal disease. Various approaches are being investigated to target defective pathways/molecules in this disease. However, effective therapy is still lacking. Development of specific target-based drugs for JMML remains a big challenge and represents a promising direction in this field.

Capillary nano-immunoassay for Akt 1/2/3 and 4EBP1 phosphorylation in acute myeloid leukemia

BackgroundOverall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse being a major cause of mortality. The PI3K-Akt-mTOR kinase pathway plays a vital role in pro-survival signals within leukemic cells and inhibition of this pathway is being investigated to improve patient outcomes. Tracking activation of multiple signaling proteins simultaneously in patient samples can be challenging especially with limiting cell numbers within rare sub-populations.MethodsThe NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have utilized this nano-immunoassay to examine activation of Akt 1/2/3 and downstream mTOR target - eukaryotic initiation factor 4E-Binding Protein 1 (4EBP1).ResultsAssays for Akt 1/2/3 and 4EBP1 were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. Target inhibition was studied using mTOR 1/2 inhibitor AZD-8055 and results were corroborated by Western blotting. The assay was able to quantify nanogram amounts of 4EBP1 and Akt 1/2/3 in AML cell lines and primary pediatric AML samples and results were quantifiable, consistent and reproducible.ConclusionOur data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this nano-immunoassay prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy.

Targeting MYC Dependence by Metabolic Inhibitors in Cancer.

MYC is a critical growth regulatory gene that is commonly overexpressed in a wide range of cancers. Therapeutic targeting of MYC transcriptional activity has long been a goal, but it has been difficult to achieve with drugs that directly block its DNA-binding ability. Additional approaches that exploit oncogene addiction are promising strategies against MYC-driven cancers. Also, drugs that target metabolic regulatory pathways and enzymes have potential for indirectly reducing MYC levels. Glucose metabolism and oxidative phosphorylation, which can be targeted by multiple agents, promote cell growth and MYC expression. Likewise, modulation of the signaling pathways and protein synthesis regulated by adenosine monophosphate-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) can also be an effective route for suppressing MYC translation. Furthermore, recent data suggest that metabolism of nucleotides, fatty acids and glutamine are exploited to alter MYC levels. Combination therapies offer potential new approaches to overcome metabolic plasticity caused by single agents. Although potential toxicities must be carefully controlled, new inhibitors currently being tested in clinical trials offer significant promise. Therefore, as both a downstream target of metabolism and an upstream regulator, MYC is a prominent central regulator of cancer metabolism. Exploiting metabolic vulnerabilities of MYC-driven cancers is an emerging research area with translational potential.

Synergistic cell death in FLT3-ITD positive acute myeloid leukemia by combined treatment with metformin and 6-benzylthioinosine

Current therapy for acute myeloid leukemia (AML) primarily includes high-dose cytotoxic chemotherapy with or without allogeneic stem cell transplantation. Targeting unique cellular metabolism of cancer cells is a potentially less toxic approach. Monotherapy with mitochondrial inhibitors like metformin have met with limited success since escape mechanisms such as increased glycolytic ATP production, especially in hyperglycemia, can overcome the metabolic blockade. As an alternative strategy for metformin therapy, we hypothesized that the combination of 6-benzylthioinosine (6-BT), a broad-spectrum metabolic inhibitor, and metformin could block this drug resistance mechanism. Metformin treatment alone resulted in significant suppression of ROS and mitochondrial respiration with increased glycolysis accompanied by modest cytotoxicity (10-25%). In contrast, 6-BT monotherapy resulted in inhibition of glucose uptake, decreased glycolysis, and decreased ATP with minimal changes in ROS and mitochondrial respiration. The combination of 6-BT with metformin resulted in significant cytotoxicity (60-70%) in monocytic AML cell lines and was associated with inhibition of FLT3-ITD activated STAT5 and reduced c-Myc and GLUT-1 expression. Therefore, although the anti-tumor and metabolic effects of metformin have been limited by the metabolic reprogramming within cells, the novel combination of 6-BT and metformin targets this bypass mechanism resulting in reduced glycolysis, STAT5 inhibition, and increased cell death.

Mitoxantrone as a substitute for daunorubicin during induction in newly diagnosed lymphoblastic leukemia and lymphoma

Daunorubicin, a component of the four‐drug induction chemotherapy regimen for de novo pediatric high‐risk acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LLy), was unavailable in 2011 due to a national drug shortage. During this time, our institution substituted mitoxantrone 6.25 mg/m2 for daunorubicin 25 mg/m2 on induction Days 1, 8, 15, and 22. While mitoxantrone has been shown to be effective for relapsed ALL, it has not been studied in de novo pediatric ALL/LLy.

Nanoproteomic Assays on Hematopoietic Stem Cells

Dysregulation of cytokine signaling pathways is associated with benign and malignant hematologic disorders. Improvements in therapy rely on understanding the biology of the pathways and the proteins involved. Studying these pathways in patient samples is challenging as samples are difficult to obtain, contain fewer cells, and are heterogeneous in nature. To address some of these difficulties, we have utilized the technique of microcapillary electrophoresis. Using the NanoPro 1000 system (ProteinSimple) which is built on an automated, capillary-based immunoassay platform, we have developed rapid and quantitative assays for specific proteins from relatively small sample sizes. The NanoPro provides precise and quantitative data of the phosphorylation states of a specific protein of interest. We describe our experience with NanoPro assay development and optimization with specific application toward understanding aberrant cytokine signaling in human leukemia cells.

Early T-Cell Precursor Acute Lymphoblastic Leukemia in an Infant With an NRAS Q61R Mutation and Clinical Features of Juvenile Myelomonocytic Leukemia.

Early T‐cell precursor acute lymphoblastic leukemia (ETP‐ALL) is a subtype of T‐acute lymphoblastic leukemia (T‐ALL) arising from a primitive precursor. We present a unique case of an infant with ETP‐ALL with a missense NRAS mutation in codon 61 (c.182A>G, p.Q61R). The patient also had a minor population of non‐ETP T‐ALL blasts and clinical features typically associated with juvenile myelomonocytic leukemia (JMML), namely, absolute monocytosis, splenomegaly, and elevated hemoglobin F. The treatment was initiated with chemotherapy, followed by cord blood transplantation. The patient achieved remission, but unfortunately died from transplant‐related complications. This case highlights an NRAS mutation in ETP‐ALL with JMML‐like phenotype.

Abstract C200: Rapid nano-immunoassay for evaluation of mTOR pathway activation in acute myeloid leukemia.

Overall cure rates in acute myeloid leukemia (AML) continue to range between 60-65% with disease relapse in AML being a major cause of mortality. Risk stratification by cytogenetic and molecular aberrations has changed the prognosis for some patients, but for a majority of the patients (especially with normal cytogenetics) improvement in therapy is needed. Understanding key differences in survival pathways such as the mTOR pathway within leukemic cells could be potentially exploited to develop new drug therapy and design relapse therapy. Tracking pathway activation in patient samples is challenging in AML since samples are difficult to obtain, contain fewer cells, and are heterogeneous in nature. The NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay platform and enables a rapid and quantitative analysis of specific proteins and their phosphorylation states. We have used this novel nano-technology based system to study the mTOR pathway in AML samples (n=8) obtained from the Children9s Oncology Group (COG) Myeloid Disease Reference Laboratory. Assays for downstream mTOR target protein-eukaryotic initiation factor 4E binding protein 1 (4EBP1) were standardized using AML cell lines (MV4-11, MOLM-14, OCI-AML3 and HL-60) prior to testing in patient samples. The assay was able to detect as little as 80 ng of protein which corresponded to 1000 cells per assay. MV4-11 cells were treated with the dual mTOR 1/2 inhibitor AZD-8055 for 24 hours and reduced phosphorylation of 4EBP1 was confirmed by Western Blotting and corroborated by the NanoPro assay. This nano-immunoassay was able to reliably quantify amounts of 4EBP1 (total and phosphorylated forms) in primary pediatric AML samples (n=8) thawed following cryopreservation. The relative luminescence units (measure of chemiluminescence and protein signal) were plotted against exposure time and the linearity of the graph provided evidence that the signal is sensitive over a range of three logs. Further, these samples were treated with AZD-8055 for 24 hours followed by analysis of changes in signaling profiles for total and phosphorylated Ser65 and Thr37/46. As expected, complete reduction of phosphorylation was noted in the treated samples (n=4). The assays obtained were quantifiable, consistent, and reproducible and enabled us to delineate activation patterns of the mTOR pathway in primary AML samples and evaluate drug efficacy for targeted agents. Our data provides a strong basis for testing this platform on a larger scale and our long term aim is to utilize this novel nano-immunoassay based platform prospectively in de-novo AML to be able to identify poor responders who might benefit from early introduction of targeted therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C200. Citation Format: Himalee Sabnis, Heath Bradley, Todd Cooper, Kevin Bunting. Rapid nano-immunoassay for evaluation of mTOR pathway activation in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C200.