Generation of Induced Pluripotent Stem Cells from a Female Patient with a Xq27.3-q28 Deletion to Establish Disease Models and Identify Therapies.

Since it is extremely difficult to establish an animal model for human chromosomal abnormalities, induced pluripotent stem cells (iPSCs) provide a powerful alternative to study underlying mechanisms of these disorders and identify potential therapeutic interventions. In this study we established iPSCs from a young girl with a hemizygous deletion of Xq27.3-q28 who exhibited global developmental delay and intellectual disability from early in infancy. The deletion site on the X chromosome includes Fragile X Mental Retardation 1 (FMR1), the gene responsible for fragile X syndrome, which likely contributes to the patient's neurodevelopmental abnormalities. The FMR1 gene was expressed in approximately half of the iPSC clones we generated while it was absent in the other half due to the random inactivation of normal and abnormal X chromosomes. The normal or absent expression pattern of the FMR1 gene was not altered when the iPSCs were differentiated into neural progenitor cells (NPCs). Moreover, chromosome reactivating reagents such as 5-aza-2-deoxycytidine, trichostatin A, and UNC0638, were tested in an attempt to reactivate the suppressed FMR1 gene in affected iPSC-NPCs. The affected and control isogenic iPSCs developed in this study are ideal models with which to identify downstream consequences caused by the Xq27.3-q28 deletion and also to provide tools for high-throughput screening to identify compounds potentially improving the well-being of this patient population.

A genomics-informed computational biology platform prospectively predicts treatment responses in AML and MDS patients.

Patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) are generally older and have more comorbidities. Therefore, identifying personalized treatment options for each patient early and accurately is essential. To address this, we developed a computational biology modeling (CBM) and digital drug simulation platform that relies on somatic gene mutations and gene CNVs found in malignant cells of individual patients. Drug treatment simulations based on unique patient-specific disease networks were used to generate treatment predictions. To evaluate the accuracy of the genomics-informed computational platform, we conducted a pilot prospective clinical study (NCT02435550) enrolling confirmed MDS and AML patients. Blinded to the empirically prescribed treatment regimen for each patient, genomic data from 50 evaluable patients were analyzed by CBM to predict patient-specific treatment responses. CBM accurately predicted treatment responses in 55 of 61 (90%) simulations, with 33 of 61 true positives, 22 of 61 true negatives, 3 of 61 false positives, and 3 of 61 false negatives, resulting in a sensitivity of 94%, a specificity of 88%, and an accuracy of 90%. Laboratory validation further confirmed the accuracy of CBM-predicted activated protein networks in 17 of 19 (89%) samples from 11 patients. Somatic mutations in the TET2, IDH1/2, ASXL1, and EZH2 genes were discovered to be highly informative of MDS response to hypomethylating agents. In sum, analyses of patient cancer genomics using the CBM platform can be used to predict precision treatment responses in MDS and AML patients.

Computational modeling of early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) to identify personalized therapy using genomics.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive hematological malignancy for which optimal therapeutic approaches are poorly characterized. Using computational biology modeling (CBM) in conjunction with genomic data from cell lines and individual patients, we generated disease-specific protein network maps that were used to identify unique characteristics associated with the mutational profiles of ETP-ALL compared to non-ETP-ALL (T-ALL) cases and simulated cellular responses to a digital library of FDA-approved and investigational agents. Genomics-based classification of ETP-ALL patients using CBM had a prediction sensitivity and specificity of 93% and 87%, respectively. This analysis identified key genomic and pathway characteristics that are distinct in ETP-ALL including deletion of nucleophosmin-1 (NPM1), mutations of which are used to direct therapeutic decisions in acute myeloid leukemia. Computational simulations based on mutational profiles of 62 ETP-ALL patient models identified 87 unique targeted combination therapies in 56 of the 62 patients despite actionable mutations being present in only 37% of ETP-ALL patients. Shortlisted two-drug combinations were predicted to be synergistic in 11 profiles and were validated by in vitro chemosensitivity assays. In conclusion, computational modeling was able to identify unique biomarkers and pathways for ETP-ALL, and identify new drug combinations for potential clinical testing.

Exploring the Structure-Activity Relationship and Mechanism of a Chromene Scaffold (CXL Series) for Its Selective Antiproliferative Activity toward Multidrug-Resistant Cancer Cells.

Multidrug resistance (MDR) is one major barrier in cancer management, which urges for new drugs to help treat MDR malignancies and elucidate MDR mechanisms. A series of chromene compounds (the CXL series) demonstrate increased antiproliferative activity toward MDR acute-myeloid-leukemia (AML) cells. The structure-activity relationship (SAR) of the antiproliferative potency has been partly characterized, whereas the structural determinants contributing to selectivity have not been investigated. In this study, three series of CXL compounds were synthesized and evaluated in HL60 and HL60/MX2 leukemia cells. The results not only confirmed previous SAR studies but also, for the first time, provided structural insights into the selectivity for MDR HL60/MX2 cells. Using the lead compounds as probes, we demonstrated that their modulation of intracellular-calcium homeostasis results in their antiproliferative potency and selectivity. Three candidates also demonstrate excellent in vitro safety profiles between cancer cells and normal cells, which will be evaluated in vivo in future studies.

Ex vivo virotherapy with myxoma virus does not impair hematopoietic stem and progenitor cells.

BACKGROUND: Relapsing disease is a major challenge after hematopoietic cell transplantation for hematological malignancies. Myxoma virus (MYXV) is an oncolytic virus that can target and eliminate contaminating cancer cells from auto-transplant grafts. The aims of this study were to examine the impact of MYXV on normal hematopoietic stem and progenitor cells and define the optimal treatment conditions for ex vivo virotherapy. METHODS: Bone marrow (BM) and mobilized peripheral blood stem cells (mPBSCs) from patients with hematologic malignancies were treated with MYXV at various time, temperature and incubation media conditions. Treated BM cells from healthy normal donors were evaluated using flow cytometry for MYXV infection, long-term culture-initiating cell (LTC-IC) assay and colony-forming cell (CFC) assay. RESULTS: MYXV initiated infection in up to 45% of antigen-presenting monocytes, B cells and natural killer cells; however, these infections were uniformly aborted in >95% of all cells. Fresh graft sources showed higher levels of MYXV infection initiation than cryopreserved specimens, but in all cases less than 10% of CD34(+) cells could be infected after ex vivo MYXV treatment. MYXV did not impair LTC-IC colony numbers compared with mock treatment. CFC colony types and numbers were also not impaired by MYXV treatment. MYXV incubation time, temperature or culture media did not significantly change the percentage of infected cells, LTC-IC colony formation or CFC colony formation. CONCLUSIONS: Human hematopoietic cells are non-permissive for MYXV. Human hematopoietic stem and progenitor cells were not infected and thus unaffected by MYXV ex vivo treatment.

Chemosensitizing AML cells by targeting bone marrow endothelial cells.

Refractory disease is the greatest challenge in treating patients with acute myeloid leukemia (AML). Blood vessels may serve as sanctuary sites for AML. When AML cells were co-cultured with bone marrow endothelial cells (BMECs), a greater proportion of leukemia cells were in G0/G1. This led us to a strategy of targeting BMECs with tubulin-binding combretastatins, causing BMECs to lose their flat phenotype, degrade their cytoskeleton, cease growth, and impair migration despite unchanged BMEC viability and metabolism. Combretastatins also caused downregulation of BMEC adhesion molecules known to tether AML cells, including vascular cell adhesion molecule (VCAM)-1 and vascular endothelial (VE)-cadherin. When AML-BMEC co-cultures were treated with combretastatins, a significantly greater proportion of AML cells dislodged from BMECs and entered the G2/M cell cycle, suggesting enhanced susceptibility to cell cycle agents. Indeed, the combination of combretastatins and cytotoxic chemotherapy enhanced additive AML cell death. In vivo mice xenograft studies confirmed this finding by revealing complete AML regression after treatment with combretastatins and cytotoxic chemotherapy. Beyond highlighting the pathologic role of BMECs in the leukemia microenvironment as a protective reservoir of disease, these results support a new strategy for using vascular-targeting combretastatins in combination with cytotoxic chemotherapy to treat AML.

Endothelial cell derived angiocrine support of acute myeloid leukemia targeted by receptor tyrosine kinase inhibition.

In acute myeloid leukemia (AML), refractory disease is a major challenge and the leukemia microenvironment may harbor refractory disease. Human AML cell lines KG-1 and HL-60 expressed receptors also found on endothelial cells (ECs) such as VEGFRs, PDGFRs, and cKit. When human AML cells were co-cultured with human umbilical vein endothelial cells (HUVECs) and primary bone marrow endothelial cell (BMECs), the AML cells were more resistant to cytarabine chemotherapy, even in transwell co-culture suggesting angiocrine regulation. Primary BMECs secreted significantly increased levels of VEGF-A and PDGF-AB after exposure to cytarabine. Pazopanib, a receptor tyrosine kinase inhibitor (RTKI) of VEGFRs, PDGFRs, and cKit, removed EC protection of AML cells and enhanced AML cell sensitivity to cytarabine. Xenograft modeling showed significant regression of AML cells and abrogation of BM hypervascularity in RTKI treated cohorts. Together, these results show direct cytotoxicity of RTKIs on AML cells and reversal of EC protection. Combining RTKIs with chemotherapy may serve as promising therapeutic strategy for patients with AML.

Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells.

Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematologic malignancies, but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally, strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently, using a xenograft model, we serendipitously discovered that myxoma virus (MYXV) prevented GVHD while permitting GVM. In this study, we show that MYXV binds to resting, primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-γ, interleukin-2 (IL-2), and soluble IL-2Rα, but did not affect expression of IL-4 and IL-10. MYXV suppressed T-cell proliferation in 2 patterns (full vs partial) depending on the donor. In terms of GVM, we show that MYXV-infected activated human T lymphocytes effectively deliver live oncolytic virus to human multiple myeloma cells, thus augmenting GVM by transfer of active oncolytic virus to residual cancer cells. Given this dual capacity of reducing GVHD plus increasing the antineoplastic effectiveness of GVM, ex vivo virotherapy with MYXV may be a promising clinical adjunct to allo-HCT regimens.

Detailed analysis of bone marrow from patients with ischemic heart disease and left ventricular dysfunction: BM CD34, CD11b, and clonogenic capacity as biomarkers for clinical outcomes.

RATIONALE: Bone marrow (BM) cell therapy for ischemic heart disease (IHD) has shown mixed results. Before the full potency of BM cell therapy can be realized, it is essential to understand the BM niche after acute myocardial infarction (AMI). OBJECTIVE: To study the BM composition in patients with IHD and severe left ventricular (LV) dysfunction. METHODS AND RESULTS: BM from 280 patients with IHD and LV dysfunction were analyzed for cell subsets by flow cytometry and colony assays. BM CD34(+) cell percentage was decreased 7 days after AMI (mean of 1.9% versus 2.3%-2.7% in other cohorts; P<0.05). BM-derived endothelial colonies were significantly decreased (P<0.05). Increased BM CD11b(+) cells associated with worse LV ejection fraction (LVEF) after AMI (P<0.05). Increased BM CD34(+) percentage associated with greater improvement in LVEF (+9.9% versus +2.3%; P=0.03, for patients with AMI and +6.6% versus -0.02%; P=0.021 for patients with chronic IHD). In addition, decreased BM CD34(+) percentage in patients with chronic IHD correlated with decrement in LVEF (-2.9% versus +0.7%; P=0.0355). CONCLUSIONS: In this study, we show a heterogeneous mixture of BM cell subsets, decreased endothelial colony capacity, a CD34+ cell nadir 7 days after AMI, a negative correlation between CD11b percentage and postinfarct LVEF, and positive correlation of CD34 percentage with change in LVEF after cell therapy. These results serve as a possible basis for the small clinical improvement seen in autologous BM cell therapy trials and support selection of potent cell subsets and reversal of comorbid BM impairment. CLINICAL TRIAL REGISTRATIONS URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00684021, NCT00684060, and NCT00824005.

Functional integration of acute myeloid leukemia into the vascular niche.

Vascular endothelial cells are a critical component of the hematopoietic microenvironment that regulates blood cell production. Recent studies suggest the existence of functional cross-talk between hematologic malignancies and vascular endothelium. Here we show that human acute myeloid leukemia (AML) localizes to the vasculature in both patients and in a xenograft model. A significant number of vascular tissue-associated AML cells (V-AML) integrate into vasculature in vivo and can fuse with endothelial cells. V-AML cells acquire several endothelial cell-like characteristics, including the upregulation of CD105, a receptor associated with activated endothelium. Remarkably, endothelial-integrated V-AML shows an almost fourfold reduction in proliferative activity compared with non-vascular-associated AML. Primary AML cells can be induced to downregulate the expression of their hematopoietic markers in vitro and differentiate into phenotypically and functionally defined endothelial-like cells. After transplantation, these leukemia-derived endothelial cells are capable of giving rise to AML. These novel functional interactions between AML cells and normal endothelium along with the reversible endothelial cell potential of AML suggest that vascular endothelium may serve as a previously unrecognized reservoir for AML.

Virotherapy using myxoma virus prevents lethal graft-versus-host disease following xeno-transplantation with primary human hematopoietic stem cells.

Graft-versus-host disease (GVHD) is a potentially lethal clinical complication arising from the transfer of alloreactive T lymphocytes into immunocompromised recipients. Despite conventional methods of T cell depletion, GVHD remains a major challenge in allogeneic hematopoietic cell transplant. Here, we demonstrate a novel method of preventing GVHD by ex vivo treatment of primary human hematopoietic cell sources with myxoma virus, a rabbit specific poxvirus currently under development for oncolytic virotherapy. This pretreatment dramatically increases post-transplant survival of immunocompromised mice injected with primary human bone marrow or peripheral blood cells and prevents the expansion of human CD3(+) lymphocytes in major recipient organs. Similar viral treatment also prevents human-human mixed alloreactive T lymphocyte reactions in vitro. Our data suggest that ex vivo virotherapy with myxoma virus can be a simple and effective method for preventing GVHD following infusion of hematopoietic products containing alloreactive T lymphocytes such as: allogeneic hematopoietic stem and progenitor cells, donor leukocyte infusions and blood transfusions.

Acute myeloid leukemia targeting by myxoma virus in vivo depends on cell binding but not permissiveness to infection in vitro.

Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo.

Leukemia regression by vascular disruption and antiangiogenic therapy.

Acute myelogenous leukemias (AMLs) and endothelial cells depend on each other for survival and proliferation. Monotherapy antivascular strategies such as targeting vascular endothelial growth factor (VEGF) has limited efficacy in treating AML. Thus, in search of a multitarget antivascular treatment strategy for AML, we tested a novel vascular disrupting agent, OXi4503, alone and in combination with the anti-VEGF antibody, bevacizumab. Using xenotransplant animal models, OXi4503 treatment of human AML chloromas led to vascular disruption in leukemia cores that displayed increased leukemia cell apoptosis. However, viable rims of leukemia cells remained and were richly vascular with increased VEGF-A expression. To target this peripheral reactive angiogenesis, bevacizumab was combined with OXi4503 and abrogated viable vascular rims, thereby leading to enhanced leukemia regression. In a systemic model of primary human AML, OXi4503 regressed leukemia engraftment alone and in combination with bevacizumab. Differences in blood vessel density alone could not account for the observed regression, suggesting that OXi4503 also exhibited direct cytotoxic effects on leukemia cells. In vitro analyses confirmed this targeted effect, which was mediated by the production of reactive oxygen species and resulted in apoptosis. Together, these data show that OXi4503 alone is capable of regressing AML by a multitargeted mechanism and that the addition of bevacizumab mitigates reactive angiogenesis.

De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2'-deoxycytidine.

The deoxycytidine analog 5-aza-2'-deoxycitidine (5-aza-dC) is a potent chemotherapeutic agent effective against selective types of cancer. The molecular mechanism by which 5-aza-dC induces cancer cell death, however, is not fully understood. It has been accepted that the mechanism of toxicity is due to the covalent binding between the DNA methyltransferase (Dnmt) and 5-aza-dC-substituted DNA. In order to define which member of the Dnmt family plays a dominant role in the cytotoxicity, we examined the effect of 5-aza-dC on cell growth and apoptosis in various Dnmt null mutant embryonic stem (ES) cells. Of interest, Dnmt3a-Dnmt3b double null ES cells were highly resistant to 5-aza-dC when compared to wild type, Dnmt3a null, Dnmt3b null, or Dnmt1 null ES cells. The cellular sensitivity to 5-aza-dC correlated well with the expression status of Dnmt3 in both undifferentiated and differentiated ES cells. When exogenous Dnmt3a or Dnmt3b was expressed in double null ES cells, the sensitivity to 5-aza-dC was partially restored. These results suggest that the cytotoxic effect of 5-aza-dC may be mediated primarily through Dnmt3a and Dnmt3b de novo DNA methyltransferases. Further, the ability to form Dnmt-DNA adducts was similar in Dnmt1 and Dnmt3, and the expression level of Dnmt3 was not higher than that of Dnmt1 in ES cells. Therefore, Dnmt3-DNA adducts may be more effective for inducing apoptosis than Dnmt1-DNA adducts. These results imply a therapeutic potential of 5-aza-dC to cancers expressing Dnmt3.