Sanket S. Acharya

AKT/FOXO Signaling Enforces Reversible Differentiation Blockade in Myeloid Leukemias

AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in ∼40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions.

Circulating miR-29a and miR-150 correlate with delivered dose during thoracic radiation therapy for non-small cell lung cancer

BackgroundRisk of normal tissue toxicity limits the amount of thoracic radiation therapy (RT) that can be routinely prescribed to treat non-small cell lung cancer (NSCLC). An early biomarker of response to thoracic RT may provide a way to predict eventual toxicities—such as radiation pneumonitis—during treatment, thereby enabling dose adjustment before the symptomatic onset of late effects. MicroRNAs (miRNAs) were studied as potential serological biomarkers for thoracic RT. As a first step, we sought to identify miRNAs that correlate with delivered dose and standard dosimetric factors.MethodsWe performed miRNA profiling of plasma samples obtained from five patients with Stage IIIA NSCLC at five dose-points each during radical thoracic RT. Candidate miRNAs were then assessed in samples from a separate cohort of 21 NSCLC patients receiving radical thoracic RT. To identify a cellular source of circulating miRNAs, we quantified in vitro miRNA expression intracellularly and within secreted exosomes in five NSCLC and stromal cell lines.ResultsmiRNA profiling of the discovery cohort identified ten circulating miRNAs that correlated with delivered RT dose as well as other dosimetric parameters such as lung V20. In the validation cohort, miR-29a-3p and miR-150-5p were reproducibly shown to decrease with increasing radiation dose. Expression of miR-29a-3p and miR-150-5p in secreted exosomes decreased with radiation. This was concomitant with an increase in intracellular levels, suggesting that exosomal export of these miRNAs may be downregulated in both NSCLC and stromal cells in response to radiation.ConclusionsmiR-29a-3p and miR-150-5p were identified as circulating biomarkers that correlated with delivered RT dose. miR-150 has been reported to decrease in the circulation of mammals exposed to radiation while miR-29a has been associated with fibrosis in the human heart, lungs, and kidneys. One may therefore hypothesize that outlier levels of circulating miR-29a-3p and miR-150-5p may eventually help predict unexpected responses to radiation therapy, such as toxicity.

Serum microRNAs are early indicators of survival after radiation-induced hematopoietic injury.

Serum miRNAs can predict long-term radiation-induced hematopoietic injury immediately after radiation and thereby facilitate timely medical intervention and improve overall survival of exposed individuals. Spotting radiation injury with serum microRNAs Three Mile Island, Chernobyl, and Fukushima were catastrophic nuclear power plant accidents in the United States, Ukraine, and Japan, respectively. The radiation from these accidents took terrible tolls on human lives, not only at the time of the accident, but also long-term, as individuals suffer from unpredictable cancer, gut damage, and infections. Predicting such toxicity is imperfect, and current methods do not account for latent damage to organs and systems. In a crucial step toward better indicators of radiation injury, Acharya and colleagues investigated microRNA profiles and hematopoietic damage in mice exposed to various doses of total body irradiation (TBI). Mice received between 0 and 8 Gy TBI. Serum miRNA profiles distinguished animals receiving different doses of radiation, whereas bone marrow mononuclear cell counts did not. Such miRNA signatures may be useful in distinguishing humans with mild radiation-related injury from those with more severe (often nonrecoverable) bone marrow damage—even if all were exposed to sublethal doses of TBI. Importantly, animals receiving radiation mitigation in the form of amifostine, a radioprotectant, or stem cell transplant, demonstrated serum miRNA profiles that changed to match 0-Gy controls, indicating that miRNAs reflect impact of radiation (hematopoietic function) rather than dose. Similar results were obtained in “humanized” mice, suggesting the translatability of this miRNA-based approach to predicting radiation toxicity in people. Future studies with human samples will allow for validation of such indicators, as well as further investigations into novel intervention measures, to improve care of patients and enhance survival after radiation exposure. Accidental radiation exposure is a threat to human health that necessitates effective clinical planning and diagnosis. Minimally invasive biomarkers that can predict long-term radiation injury are urgently needed for optimal management after a radiation accident. We have identified serum microRNA (miRNA) signatures that indicate long-term impact of total body irradiation (TBI) in mice when measured within 24 hours of exposure. Impact of TBI on the hematopoietic system was systematically assessed to determine a correlation of residual hematopoietic stem cells (HSCs) with increasing doses of radiation. Serum miRNA signatures distinguished untreated mice from animals exposed to radiation and correlated with the impact of radiation on HSCs. Mice exposed to sublethal (6.5 Gy) and lethal (8 Gy) doses of radiation were indistinguishable for 3 to 4 weeks after exposure. A serum miRNA signature detectable 24 hours after radiation exposure consistently segregated these two cohorts. Furthermore, using either a radioprotective agent before, or radiation mitigation after, lethal radiation, we determined that the serum miRNA signature correlated with the impact of radiation on animal health rather than the radiation dose. Last, using humanized mice that had been engrafted with human CD34+ HSCs, we determined that the serum miRNA signature indicated radiation-induced injury to the human bone marrow cells. Our data suggest that serum miRNAs can serve as functional dosimeters of radiation, representing a potential breakthrough in early assessment of radiation-induced hematopoietic damage and timely use of medical countermeasures to mitigate the long-term impact of radiation.

Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning.

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions.