FLT3-ITD impedes retinoic acid, but not arsenic, responses in murine acute promyelocytic leukemias.

Acute promyelocytic leukemia (APL) is often associated with activating FLT3 signaling mutations. These are highly related to hyperleukocytosis, a major adverse risk factor with chemotherapy-based regimens. APL is a model for oncogene-targeted therapies: all-trans retinoic acid (ATRA) and arsenic both target and degrade its ProMyelocytic Leukemia/Retinoic Acid Receptor α (PML/RARA) driver. The combined ATRA/arsenic regimen now cures virtually all patients with standard-risk APL. Although FLT3-internal tandem duplication (ITD) was an adverse risk factor for historical ATRA/chemotherapy regimens, the molecular bases for this effect remain unknown. Using mouse APL models, we unexpectedly demonstrate that FLT3-ITD severely blunts ATRA response. Remarkably, although the transcriptional output of initial ATRA response is unaffected, ATRA-induced PML/RARA degradation is blunted, as is PML nuclear body reformation and activation of P53 signaling. Critically, the combination of ATRA and arsenic fully rescues therapeutic response in FLT3-ITD APLs, restoring PML/RARA degradation, PML nuclear body reformation, P53 activation, and APL eradication. Moreover, arsenic targeting of normal PML also contributes to APL response in vivo. These unexpected results explain the less favorable outcome of FLT3-ITD APLs with ATRA-based regimens, and stress the key role of PML nuclear bodies in APL eradication by the ATRA/arsenic combination.

Outcomes at 7 years post-transplant in black vs nonblack kidney transplant recipients administered belatacept or cyclosporine in BENEFIT and BENEFIT-EXT.

Clinical outcomes are generally worse for black vs nonblack renal allograft recipients. In BENEFIT and BENEFIT-EXT, recipients were randomized to belatacept more intense-based, belatacept less intense-based, or cyclosporine-based immunosuppression. At year 7, belatacept was associated with superior graft survival vs cyclosporine in BENEFIT (recipients of living or standard criteria deceased donor kidneys); belatacept was associated with similar graft survival vs cyclosporine in BENEFIT-EXT (recipients of extended criteria donor kidneys). In both studies, renal function was superior for belatacept-treated vs cyclosporine-treated patients. Seven-year outcomes were examined by race post hoc in each study. The effect of race and treatment on time to death or graft loss was compared using Cox regression. The interaction between treatment and race was also considered. Glomerular filtration rate (GFR) was estimated from months 1 to 84 using a repeated-measures model. In total, 8.3% (55/666) and 13.1% (71/543) of patients in BENEFIT and BENEFIT-EXT, respectively, were black. Time to death or graft loss was similar in blacks and nonblacks. For both subgroups, estimated mean GFR increased over 7 years for belatacept, but declined for cyclosporine. Outcomes were similar in belatacept-treated black and nonblack patients. Due to the small number of black patients, these results must be interpreted with caution.

RGTA® or ReGeneraTing Agents mimic heparan sulfate in regenerative medicine: from concept to curing patients.

The importance of extracellular matrix (ECM) integrity in maintaining normal tissue function is highlighted by numerous pathologies and situations of acute and chronic injury associated with dysregulation or destruction of ECM components. Heparan sulfate (HS) is a key component of the ECM, where it fulfils important functions associated with tissue homeostasis. Its degradation following tissue injury disrupts this delicate equilibrium and may impair the wound healing process. ReGeneraTing Agents (RGTA®s) are polysaccharides specifically designed to replace degraded HS in injured tissues. The unique properties of RGTA® (resistance to degradation, binding and protection of ECM structural and signaling proteins, like HS) permit the reconstruction of the ECM, restoring both structural and biochemical functions to this essential substrate, and facilitating the processes of tissue repair and regeneration. Here, we review 25 years of research surrounding this HS mimic, supporting the mode of action, pre-clinical studies and therapeutic efficacy of RGTA® in the clinic, and discuss the potential of RGTA® in new branches of regenerative medicine.

Routine ultrasonography surveillance of native kidneys for renal cell carcinoma in kidney transplant candidates.

Renal cell carcinoma (RCC) has a high incidence in the kidney transplant population and annual surveillance detects these tumors early in their natural history. Minimal guidelines exist regarding RCC surveillance in ESRD patients awaiting transplant. A retrospective review of our kidney transplant database examined the outcomes of annual ultrasonographic surveillance during initial kidney transplant evaluation and upon annual reassessment. Of 2642 patients listed for transplant, 145 patients were found to have masses during initial kidney transplant evaluation or annual imaging consistent with new complex cystic disease or RCC. A total of 71 patients had RCC identified, with 52 found on initial kidney transplant evaluation and 19 identified on annual surveillance. Male gender and African-American race were independently associated with RCC (P<.05). RCC was detected a median of 2.0 years after listing (two annual ultrasonography studies). Patients with complex cysts were more likely to undergo transplantation (48.7%) compared to patients with RCC (21.1%; P<.001). There was no significant difference in survival between RCC patients and those found to have complex cystic disease, suggesting incidental RCC can be diagnosed early in the natural history and at a curable stage through implementation of a biennial surveillance program.

Belatacept and Long-Term Outcomes in Kidney Transplantation.

BACKGROUND: In previous analyses of BENEFIT, a phase 3 study, belatacept-based immunosuppression, as compared with cyclosporine-based immunosuppression, was associated with similar patient and graft survival and significantly improved renal function in kidney-transplant recipients. Here we present the final results from this study. METHODS: We randomly assigned kidney-transplant recipients to a more-intensive belatacept regimen, a less-intensive belatacept regimen, or a cyclosporine regimen. Efficacy and safety outcomes for all patients who underwent randomization and transplantation were analyzed at year 7 (month 84). RESULTS: A total of 666 participants were randomly assigned to a study group and underwent transplantation. Of the 660 patients who were treated, 153 of the 219 patients treated with the more-intensive belatacept regimen, 163 of the 226 treated with the less-intensive belatacept regimen, and 131 of the 215 treated with the cyclosporine regimen were followed for the full 84-month period; all available data were used in the analysis. A 43% reduction in the risk of death or graft loss was observed for both the more-intensive and the less-intensive belatacept regimens as compared with the cyclosporine regimen (hazard ratio with the more-intensive regimen, 0.57; 95% confidence interval [CI], 0.35 to 0.95; P=0.02; hazard ratio with the less-intensive regimen, 0.57; 95% CI, 0.35 to 0.94; P=0.02), with equal contributions from the lower rates of death and graft loss. The mean estimated glomerular filtration rate (eGFR) increased over the 7-year period with both belatacept regimens but declined with the cyclosporine regimen. The cumulative frequencies of serious adverse events at month 84 were similar across treatment groups. CONCLUSIONS: Seven years after transplantation, patient and graft survival and the mean eGFR were significantly higher with belatacept (both the more-intensive regimen and the less-intensive regimen) than with cyclosporine. (Funded by Bristol-Myers Squibb; ClinicalTrials.gov number, NCT00256750.).

Late presentation of adenovirus-induced hemorrhagic cystitis and ureteral obstruction in a kidney-pancreas transplant recipient.

We report a late presentation of adenovirus-induced renal allograft and bladder infection causing azotemia and hemorrhagic cystitis in a patient 5 years after simultaneous kidney-pancreas transplantation. Adenovirus has been increasingly recognized as a cause of morbidity and mortality in both solid organ and stem cell transplant recipients. We wish to emphasize the importance of early detection, as treatment options involve reduction of immunosuppression, followed by the addition of antiviral agents and supportive care.

Identification of genes associated with blood feeding in the cat flea, Ctenocephalides felis.

BACKGROUND: The cat flea (Ctenocephalides felis) is a blood-feeding ectoparasitic insect and particular nuisance pest of companion animals worldwide. Identification of genes that are differentially expressed in response to feeding is important for understanding flea biology and discovering targets for their control. METHODS: C. felis fleas were maintained and fed for 24 h using an artificial rearing system. The technique of suppression subtractive hybridization was employed to screen for mRNAs specifically expressed in fed fleas. RESULTS: We characterized nine distinct full-length flea transcripts that exhibited modulated or de novo expression during feeding. Among the predicted protein sequences were two serine proteases, a serine protease inhibitor, two mucin-like molecules, a DNA topoisomerase, an enzyme associated with GPI-mediated cell membrane attachment of proteins and a component of the insect innate immune response. CONCLUSIONS: Our results provide a molecular insight into the physiology of flea feeding. The protein products of the genes identified may play important roles during flea feeding in terms of blood meal digestion, cellular growth/repair and protection from feeding-associated stresses.

West Nile virus infection in kidney and pancreas transplant recipients in the Dallas-Fort Worth Metroplex during the 2012 Texas epidemic.

BACKGROUND: In 2012, the United States experienced one of its worst West Nile virus (WNV) epidemics, reporting 5,387 human cases and final death toll of 243. Texas was at the epicenter of the outbreak, with 1,875 reported cases and 89 deaths that year. The Texas outbreak centered mainly in the Dallas-Fort Worth area where 30 deaths were reported. We report three cases of severe WNV infection complicated by meningoencephalitis in our organ transplant population. METHODS: Clinical data were collected from chart review. Therapy and outcomes on three identified patients were reviewed and compared with previously reported cases of WNV infection in kidney/pancreas transplant recipients and the general population. RESULTS: Two recipients of kidney and one recipient of a combined kidney and pancreas transplant were treated at our center for WNV infection. All three patients presented with a rapid decline in mental status within 24 hours of admission consistent with meningoencephalitis. Diagnosis was made based on detection of WNV IgM in the serum. All patients received intravenous immunoglobulin (IVIG) therapy at 400 mg/kg × 3 to 4 doses. As a result, two patients had a full recovery, and one patient died. CONCLUSION: Transplant recipients have a higher risk of neurologic complications from WNV infection. In areas where WNV is endemic, clinicians must have a high index of suspicion when treating patients presenting with fever, headache, and confusion. Full recovery in two of three patients suggests a potential role of IVIG therapy in controlling active WNV infection, particularly in immunosuppressed patients.

Activation of a promyelocytic leukemia-tumor protein 53 axis underlies acute promyelocytic leukemia cure.

Acute promyelocytic leukemia (APL) is driven by the promyelocytic leukemia (PML)-retinoic acid receptor-α (PML-RARA) fusion protein, which interferes with nuclear receptor signaling and PML nuclear body (NB) assembly. APL is the only malignancy definitively cured by targeted therapies: retinoic acid (RA) and/or arsenic trioxide, which both trigger PML-RARA degradation through nonoverlapping pathways. Yet, the cellular and molecular determinants of treatment efficacy remain disputed. We demonstrate that a functional Pml-transformation-related protein 53 (Trp53) axis is required to eradicate leukemia-initiating cells in a mouse model of APL. Upon RA-induced PML-RARA degradation, normal Pml elicits NB reformation and induces a Trp53 response exhibiting features of senescence but not apoptosis, ultimately abrogating APL-initiating activity. Apart from triggering PML-RARA degradation, arsenic trioxide also targets normal PML to enhance NB reformation, which may explain its clinical potency, alone or with RA. This Pml-Trp53 checkpoint initiated by therapy-triggered NB restoration is specific for PML-RARA-driven APL, but not the RA-resistant promyelocytic leukemia zinc finger (PLZF)-RARA variant. Yet, as NB biogenesis is druggable, it could be therapeutically exploited in non-APL malignancies.

Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia.

Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo. Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.

Comprehensive genomic screens identify a role for PLZF-RARalpha as a positive regulator of cell proliferation via direct regulation of c-MYC.

The t(11;17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor alpha (PLZF-RARalpha) and RARalpha-PLZF. Using a combination of chromatin immunoprecipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RARalpha that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RARalpha as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RARalpha promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RARalpha binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RARalpha may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RARalpha-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RARalpha.

PLZF is a regulator of homeostatic and cytokine-induced myeloid development.

A major question in hematopoiesis is how the system maintains long-term homeostasis whereby the generation of large numbers of differentiated cells is balanced with the requirement for maintenance of progenitor pools, while remaining sufficiently flexible to respond to periods of perturbed cellular output during infection or stress. We focused on the development of the myeloid lineage and present evidence that promyelocytic leukemia zinc finger (PLZF) provides a novel function that is critical for both normal and stress-induced myelopoiesis. During homeostasis, PLZF restricts proliferation and differentiation of human cord blood-derived myeloid progenitors to maintain a balance between the progenitor and mature cell compartments. Analysis of PLZF promoter-binding sites revealed that it represses transcription factors involved in normal myeloid differentiation, including GFI-1, C/EBPalpha, and LEF-1, and induces negative regulators DUSP6 and ID2. Loss of ID2 relieves PLZF-mediated repression of differentiation identifying it as a functional target of PLZF in myelopoiesis. Furthermore, induction of ERK1/2 by myeloid cytokines, reflective of a stress response, leads to nuclear export and inactivation of PLZF, which augments mature cell production. Thus, negative regulators of differentiation can serve to maintain developmental systems in a primed state, so that their inactivation by extrinsic signals can induce proliferation and differentiation to rapidly satisfy increased demand for mature cells.

Transcriptional regulation of FHL1 by TLX1/HOX11 is dosage, cell-type and promoter context-dependent.

TLX1/HOX11 encodes an NK-like homeodomain transcription factor that is both normally required for embryonic development and aberrantly expressed in T-cell acute lymphoblastic leukemia. Previous studies have shown that TLX1 can regulate target genes including ALDH1A1 and FHL1. However, whereas ALDH1A1 is consistently regulated by TLX1, endogenous FHL1 is only induced in a proportion of fibroblast or T-cell clones stably expressing TLX1. Here, we provide an explanation for these findings by demonstrating that the induction of FHL1, but not ALDH1A1, requires a high level of TLX1 expression in NIH 3T3 cells. In luciferase reporter assays, TLX1-mediated repression rather than activation of the FHL1 gene promoter and the magnitude of this effect was strongly influenced by the cellular background. Together, these results characterize TLX1 as a dual function regulator whose activity in respect to FHL1 is critically dependent upon its cellular concentration, as well as cell type and promoter context.

Overexpression of stem cell associated ALDH1A1, a target of the leukemogenic transcription factor TLX1/HOX11, inhibits lymphopoiesis and promotes myelopoiesis in murine hematopoietic progenitors.

TLX1/HOX11 is an oncogenic transcription factor in human T-cell leukemia, however, the molecular basis for its transforming activity has remained elusive. The ALDH1A1 gene, whose product participates in retinoic acid synthesis, was previously identified as a TLX1-responsive gene. Here, we confirm regulation of ALDH1A1 transcription by TLX1 and show that ALDH1A1 can profoundly perturb murine hematopoiesis by promoting myeloid differentiation at the expense of lymphopoiesis. Together, these data demonstrate that ALDH1A1 plays a key role in normal hematopoiesis, and confirm ALDH1A1 as a TLX1 transcriptional target that may contribute to the ability of this homeoprotein to alter cell fate and induce tumor growth.

HOX deregulation in acute myeloid leukemia.

The deregulation of homeobox (HOX) genes in acute myeloid leukemia (AML) and the potential for these master regulators to perturb normal hematopoiesis is well established. To date, overexpression of HOX genes in AML has been attributed to specific chromosomal aberrations and abnormalities involving mixed-lineage leukemia (MLL), an upstream regulator of HOX genes. The finding reported in this issue of the JCI by Scholl et al. that caudal-type homeobox transcription factor 2 (CDX2), which is capable of affecting HOX gene expression during embryogenesis, is overexpressed in 90% of patients with AML and induces a transplantable AML in murine models provides an alternative mechanism for HOX-induced leukemogenesis and yields important insights into the hierarchy of HOX gene regulation in AML (see the related article beginning on page 1037).

Expression and purification of the human homeodomain oncoprotein HOX11.

HOX11 is a transcription factor belonging to the homeodomain family that is essential for spleen development during embryogenesis. It is also tumorigenic, being associated with T-cell acute lymphoblastic leukemia in children. In order to understand the functional role of HOX11 in both normal development and malignancy, protein-DNA and protein-protein interaction studies involving this factor are required. Such investigations would be facilitated by the availability of significant amounts of purified HOX11 protein. However, expression of full-length HOX11 in bacteria has been reported to be problematic owing to fusion protein instability. Here, we report the purification of human HOX11 expressed in Escherichia coli as a soluble and functional glutathione S-transferase (GST) fusion protein. In addition, a mutant version of HOX11 was produced (HOX11 Delta H3) which lacked the DNA-recognition helix (helix 3) of the homeodomain. Through a single purification procedure using glutathione-Sepharose, 2mg of the recombinant proteins were obtained per liter of bacterial culture. Notably, recombinant GST-HOX11 fusion proteins had a markedly higher stability when purified at low temperature (4 degrees C). Purification to near-homogeneity was achieved as judged by SDS-PAGE and the purified proteins were recognized by anti-HOX11 antibodies. The biological activity of the recombinant protein was verified by the specific binding of GST-HOX11, but not GST-HOX11 Delta H3, to DNA containing consensus HOX11 recognition sites.