Margaret Kasner

The diabetes gene Pdx1 regulates the transcriptional network of pancreatic endocrine progenitor cells in mice

Heterozygous mutations in the gene encoding the pancreatic homeodomain transcription factor pancreatic duodenal homeobox 1 (PDX1) are associated with maturity onset diabetes of the young, type 4 (MODY4) and type 2 diabetes. Pdx1 governs the early embryonic development of the pancreas and the later differentiation of the insulin-producing islet beta cells of the endocrine compartment. We derived a Pdx1 hypomorphic allele that reveals a role for Pdx1 in the specification of endocrine progenitors. Mice homozygous for this allele displayed a selective reduction in endocrine lineages associated with decreased numbers of endocrine progenitors and a marked reduction in levels of mRNA encoding the proendocrine transcription factor neurogenin 3 (Ngn3). During development, Pdx1 occupies an evolutionarily conserved enhancer region of Ngn3 and interacts with the transcription factor one cut homeobox 1 (Hnf6) to activate this enhancer. Furthermore, mRNA levels of all 4 members of the transcription factor network that regulates Ngn3 expression, SRY-box containing gene 9 (Sox9), Hnf6, Hnf1b, and forkhead box A2 (Foxa2), were decreased in homozygous mice. Pdx1 also occupied regulatory sequences in Foxa2 and Hnf1b. Thus, Pdx1 contributes to specification of endocrine progenitors both by regulating expression of Ngn3 directly and by participating in a cross-regulatory transcription factor network during early pancreas development. These results provide insights that may be applicable to beta cell replacement strategies involving the guided differentiation of ES cells or other progenitor cell types into the beta cell lineage, and they suggest a molecular mechanism whereby human PDX1 mutations cause diabetes.

A Phase I Study of the Mammalian Target of Rapamycin Inhibitor Sirolimus and MEC Chemotherapy in Relapsed and Refractory Acute Myelogenous Leukemia

Purpose: Inhibiting mammalian target of rapamycin (mTOR) signaling in acute myelogenous leukemia (AML) blasts and leukemic stem cells may enhance their sensitivity to cytotoxic agents. We sought to determine the safety and describe the toxicity of this approach by adding the mTOR inhibitor, sirolimus (rapamycin), to intensive AML induction chemotherapy. Experimental Design: We performed a phase I dose escalation study of sirolimus with the chemotherapy regimen MEC (mitoxantrone, etoposide, and cytarabine) in patients with relapsed, refractory, or untreated secondary AML. Results: Twenty-nine subjects received sirolimus and MEC across five dose levels. Dose-limiting toxicities were irreversible marrow aplasia and multiorgan failure. The maximum tolerated dose (MTD) of sirolimus was determined to be a 12 mg loading dose on day 1 followed by 4 mg/d on days 2 to 7, concurrent with MEC chemotherapy. Complete or partial remissions occurred in 6 (22%) of the 27 subjects who completed chemotherapy, including 3 (25%) of the 12 subjects treated at the MTD. At the MTD, measured rapamycin trough levels were within the therapeutic range for solid organ transplantation. However, direct measurement of the mTOR target p70 S6 kinase phosphorylation in marrow blasts from these subjects only showed definite target inhibition in one of five evaluable samples. Conclusions: Sirolimus and MEC is an active and feasible regimen. However, as administered in this study, the synergy between MEC and sirolimus was not confirmed. Future studies are planned with different schedules to clarify the clinical and biochemical effects of sirolimus in AML and to determine whether target inhibition predicts chemotherapy response. (Clin Cancer Res 2009;15(21):6732–9)

A 2-step approach to myeloablative haploidentical stem cell transplantation: a phase 1/2 trial performed with optimized T-cell dosing.

Studies of haploidentical hematopoietic stem cell transplantation (HSCT) have identified threshold doses of T cells below which severe GVHD is usually absent. However, little is known regarding optimal T-cell dosing as it relates to engraftment, immune reconstitution, and relapse. To begin to address this question, we developed a 2-step myeloablative approach to haploidentical HSCT in which 27 patients conditioned with total body irradiation (TBI) were given a fixed dose of donor T cells (HSCT step 1), followed by cyclophosphamide (CY) for T-cell tolerization. A CD34-selected HSC product (HSCT step 2) was infused after CY. A dose of 2 × 10(8)/kg of T cells resulted in consistent engraftment, immune reconstitution, and acceptable rates of GVHD. Cumulative incidences of grade III-IV GVHD, nonrelapse mortality (NRM), and relapse-related mortality were 7.4%, 22.2%, and 29.6%, respectively. With a follow-up of 28-56 months, the 3-year probability of overall survival for the whole cohort is 48% and 75% in patients without disease at HSCT. In the context of CY tolerization, a high, fixed dose of haploidentical T cells was associated with encouraging outcomes, especially in good-risk patients, and can serve as the basis for further exploration and optimization of this 2-step approach. This study is registered at www.clinicaltrials.gov as NCT00429143.

A Two-Step Approach to Myeloablative Haploidentical Transplantation: Low Nonrelapse Mortality and High Survival Confirmed in Patients with Earlier Stage Disease

Haploidentical hematopoietic stem cell transplantation (HSCT) is an attractive alternative donor option based on the rapid availability of an acceptable donor for most patients and decreased cost compared with costs of other alternative donor strategies. The safety of haploidentical HSCT has increased in recent years, making it ethically feasible to offer to patients with earlier stage disease. We developed a 2-step approach to haploidentical HSCT that separates the lymphoid and myeloid portions of the graft, allowing fixed T cell dosing to improve consistency in outcome comparisons. In the initial 2-step trial, the subset of patients without morphologic disease at HSCT had high rates of disease-free survival. To confirm these results, 28 additional patients without evidence of their disease were treated and are now 15 to 45 (median, 31) months past HSCT. To date, the 2-year cumulative incidence of nonrelapse mortality is 3.6%, with only 1 patient dying of nonrelapse causes, confirming the safety of this approach. Based on low regimen toxicity, the probabilities of disease-free and overall survival at 2 years are 74% and 77%, respectively, consistent with the findings in the initial trial and supporting the use of this approach in earlier stage patients lacking a matched related donor.

Update on the therapy for myelodysplastic syndrome

The myelodysplastic syndromes (MDS) are a diverse group of clonal hematopoietic stem cell disorders characterized by cytopenias. Patients have a risk of developing acute leukemia though most subcome to complications of low blood counts. Over the past decade many novel treatments have been developed and investigation of new agents is ongoing. In this article, we discuss the classification and prognostic systems that are used in MDS, the agents available for treatment of MDS as well as review supportive and palliative care options for patients who are not candidates for, or opt against, newer treatment strategies. Am. J. Hematol. 2009.

Acute myeloid leukemia patients' clinical response to idasanutlin (RG7388) is associated with pre-treatment MDM2 protein expression in leukemic blasts.

In translational research described, we investigated biomarker expression by flow cytometry for MDM2 antagonist clinical response association in relapsed/refractory AML patients treated with idasanutlin-based therapy ( [Clinicaltrials.gov][1] identifier: [NCT01773408][2] ). As MDM2 targets p53 for

A Two-Step Haploidentical Versus a Two-Step Matched Related Allogeneic Myeloablative Peripheral Blood Stem Cell Transplantation

Haploidentical stem cell transplantation (SCT) offers a transplantation option to patients who lack an HLA-matched donor. We developed a 2-step approach to myeloablative allogeneic hematopoietic stem cell transplantation for patients with haploidentical or matched related (MR) donors. In this approach, the lymphoid and myeloid portions of the graft are administered in 2 separate steps to allow fixed T cell dosing. Cyclophosphamide is used for T cell tolerization. Given a uniform conditioning regimen, graft T cell dose, and graft-versus-host disease (GVHD) prophylaxis strategy, we compared immune reconstitution and clinical outcomes in patients undergoing 2-step haploidentical versus 2-step MR SCT. We retrospectively compared data on patients undergoing a 2-step haploidentical (n = 50) or MR (n = 27) peripheral blood SCT for high-risk hematological malignancies and aplastic anemia. Both groups received myeloablative total body irradiation conditioning. Immune reconstitution data included flow cytometric assessment of T cell subsets at day 28 and 90 after SCT. Both groups showed comparable early immune recovery in all assessed T cell subsets except for the median CD3/CD8 cell count, which was higher in the MR group at day 28 compared with that in the haploidentical group. The 3-year probability of overall survival was 70% in the haploidentical group and 71% in the MR group (P = .81), while the 3-year progression-free survival was 68% in the haploidentical group and 70% in the MR group (P = .97). The 3-year cumulative incidence of nonrelapse mortality was 10% in the haploidentical group and 4% in the MR group (P = .34). The 3-year cumulative incidence of relapse was 21% in the haploidentical group and 27% in the MR group (P = .93). The 100-day cumulative incidence of overall grades II to IV acute GVHD was higher in the haploidentical group compared with that in the MR group (40% versus 8%, P < .001), whereas the grades III and IV acute GVHD was not statistically different between both groups (haploidentical, 6%; MR, 4%; P = .49). The cumulative incidence of cytomegalovirus reactivation was also higher in the haploidentical group compared to the MR group (haploidentical, 68%; MR, 19%; P < .001). There were no deaths from GVHD in either group. Using an identical conditioning regimen, graft T cell dose, and GVHD prophylaxis strategy, comparable early immune recovery and clinical outcomes were observed in the 2-step haploidentical and MR SCT recipients.

Trisomy 8 in an allogeneic stem cell transplant recipient representative of a donor-derived constitutional abnormality.

Trisomy 8 is a common cytogenetic abnormality in myeloid malignancies. It can also be present constitutionally and is associated with a wide range of phenotypes. We report a case of a 20‐year‐old woman with acute myelogenous leukemia associated with the 11q23/MLL translocation who underwent allogeneic hematopoietic stem cell transplantation (HSCT) from a healthy, unrelated 26‐year‐old female. Cytogenetics on a bone marrow biopsy and aspirate performed 71 days after transplant to evaluate pancytopenia identified trisomy 8 in 6 of 7 cells examined. The bone marrow was hypocellular but normal by morphology and flow cytometry. Fluorescent in situ hybridization (FISH) for the original 11q23/MLL translocation was negative. Chimerism analysis using multiplex polymerase chain reaction to amplify an informative short tandem repeat demonstrated 97% donor cells. These findings were confirmed by repeat bone marrow biopsies at Day 110 after transplant and 1 year after transplant. With resolution of comorbid illness, the patients peripheral blood counts recovered and remained normal at 1 year after HSCT. FISH analysis of a cryopreserved sample of the donor graft showed trisomy 8 in 120 of 200 cells examined. This represents the first reported case of a person with constitutional trisomy 8 mosaicism serving as a stem cell donor. The case illustrates the importance of identifying donor‐derived constitutional abnormalities to avoid the assumption that these cytogenetic abnormalities after HSCT are representative of malignant disease. Am. J. Hematol., 2008.

Induced differentiation of acute myeloid leukemia cells by activation of retinoid X and liver X receptors.

Use of all-trans retinoic acid (ATRA) as a differentiation agent has been limited to acute promyelocytic leukemia (APL) as non-APL leukemias are insensitive to ATRA. We recently demonstrated that the rexinoid, bexarotene, induces differentiation and therapeutic responses in patients with refractory AML. Rexinoids bind and activate retinoid X receptors (RXRs); however, rexinoids alone are incapable of activating retinoic acid receptor (RAR)/RXR complexes, suggesting that myeloid differentiation can occur independent of RAR. In this study, we demonstrate that rexinoid differentiation of AML cells is RAR independent and requires the expression of PU.1. Because of the promiscuousness of RXR with other nuclear receptors, myeloid differentiation by bexarotene with other nuclear receptor ligands was explored. Bexarotene cooperated with ATRA to enhance differentiation in some AML cell lines; however, the combination of bexarotene with the PPARγ agonist rosiglitazone did not. In contrast, bexarotene combined with liver X receptor (LXR) agonists, T0901317 or GW3965, induced potent differentiation and cytotoxicity in AML cell lines and primary human AML cells, but not in normal progenitor cells. These results suggest that RXR/LXR-regulated gene expression in normal cells is deregulated in AML cells and identifies a potential role for these agonists in differentiation therapy of non-APLs.

Polo-like kinase and its inhibitors: Ready for the match to start?

Polo‐like kinases (Plks) plays a central role in the normal cell cycle and their upregulation has been shown to play a role in the pathogenesis of multiple human cancers. Preclinical work demonstrates that targeting Plk has a significant impact on the treatment of both solid and hematologic malignancies in vitro and in vivo. We review here the basic science and clinical work to date with the Plks as well as future directions with this novel class of mitotic inhibitors. Am. J. Hematol. 90:1071–1076, 2015.