The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells
. This process requires major molecular adaptations to allow HSCs to meet ...the regulatory and metabolic requirements for cell division
. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)
, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.
Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem ...and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.
Thrombocytopenia, prevalent in the majority of patients with myeloid malignancies, such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), is an independent adverse prognostic factor. ...Azacitidine (AZA), a mainstay therapeutic agent for stem cell transplant-ineligible patients with MDS/AML, often transiently induces or further aggravates disease-associated thrombocytopenia by an unknown mechanism. Here, we uncover the critical role of an acute type-I interferon (IFN-I) signaling activation in suppressing megakaryopoiesis in AZA-mediated thrombocytopenia. We demonstrate that megakaryocytic lineage-primed progenitors present IFN-I receptors and, upon AZA exposure, engage STAT1/SOCS1-dependent downstream signaling prematurely attenuating thrombopoietin receptor (TPO-R) signaling and constraining megakaryocytic progenitor cell growth and differentiation following TPO-R stimulation. Our findings directly implicate RNA demethylation and IFN-I signal activation as a root cause for AZA-mediated thrombocytopenia and suggest mitigation of TPO-R inhibitory innate immune signaling as a suitable therapeutic strategy to support platelet production, particularly during the early phases of AZA therapy.
The terrorist attacks on the World Trade Center (WTC) created an unprecedented environmental exposure to aerosolized dust, gases and potential carcinogens. Clonal hematopoiesis (CH) is defined as the ...acquisition of somatic mutations in blood cells and is associated with smoking and exposure to genotoxic stimuli. Here we show that deep targeted sequencing of blood samples identified a significantly higher proportion of WTC-exposed first responders with CH (10%; 48 out of 481) when compared with non-WTC-exposed firefighters (6.7%; 17 out of 255; odds ratio, 3.14; 95% confidence interval, 1.64-6.03; P = 0.0006) after controlling for age, sex and race/ethnicity. The frequency of somatic mutations in WTC-exposed first responders showed an age-related increase and predominantly affected DNMT3A, TET2 and other CH-associated genes. Exposure of lymphoblastoid cells to WTC particulate matter led to dysregulation of DNA replication at common fragile sites in vitro. Moreover, mice treated with WTC particulate matter developed an increased burden of mutations in hematopoietic stem and progenitor cell compartments. In summary, the high burden of CH in WTC-exposed first responders provides a rationale for enhanced screening and preventative efforts in this population.
Cancer patients show increased morbidity with COVID-19 and need effective immunization strategies. Many healthcare regulatory agencies recommend administering 'booster' doses of COVID-19 vaccines ...beyond the standard two-dose series, for this group of patients. Therefore, studying the efficacy of these additional vaccine doses against SARS-CoV-2 and variants of concern is of utmost importance in this immunocompromised patient population.
We conducted a prospective single arm clinical trial enrolling patients with cancer that had received two doses of mRNA or one dose of AD26.CoV2.S vaccine and administered a third dose of mRNA vaccine. We further enrolled patients that had no or low responses to three mRNA COVID vaccines and assessed the efficacy of a fourth dose of mRNA vaccine. Efficacy was assessed by changes in anti-spike antibody, T-cell activity, and neutralization activity, which were again assessed at baseline and 4 weeks.
We demonstrate that a third dose of COVID-19 vaccine leads to seroconversion in 57% of patients that were seronegative after primary vaccination series. The immune response is durable as assessed by anti-SARS-CoV-2 (anti-S) antibody titers, T-cell activity, and neutralization activity against wild-type (WT) SARS-CoV2 and BA1.1.529 at 6 months of follow-up. A subset of severely immunocompromised hematologic malignancy patients that were unable to mount an adequate immune response (titer <1000 AU/mL) after the third dose and were treated with a fourth dose in a prospective clinical trial which led to adequate immune boost in 67% of patients. Low baseline IgM levels and CD19 counts were associated with inadequate seroconversion. Booster doses induced limited neutralization activity against the Omicron variant.
These results indicate that third dose of COVID vaccine induces durable immunity in cancer patients and an additional dose can further stimulate immunity in a subset of patients with inadequate response.
Leukemia Lymphoma Society, National Cancer Institute.
NCT05016622.
Cytosine hypermethylation in and around DNA-binding sites of master transcription factors, including PU.1, occurs in aging hematopoietic stem cells following acquired loss-of-function mutations of ...DNA methyl-cytosine dioxygenase ten-eleven translocation-2 (TET2), albeit functional relevance has been unclear. We show that Tet2-deficient mouse hematopoietic stem and progenitor cells undergo malignant transformation upon compromised gene regulation through heterozygous deletion of an upstream regulatory region (UREΔ/WT) of the PU.1 gene. Although compatible with multilineage blood formation at young age, Tet2-deficient PU.1 UREΔ/WT mice develop highly penetrant, transplantable acute myeloid leukemia (AML) during aging. Leukemic stem and progenitor cells show hypermethylation at putative PU.1-binding sites, fail to activate myeloid enhancers, and are hallmarked by a signature of genes with impaired expression shared with human AML. Our study demonstrates that Tet2 and PU.1 jointly suppress leukemogenesis and uncovers a methylation-sensitive PU.1-dependent gene network as a unifying molecular vulnerability associated with AML.
We identify moderately impaired PU.1 mRNA expression as a biological modality predisposing Tet2-deficient hematopoietic stem and progenitor cells to malignant transformation. Our study furthermore uncovers a methylation-sensitive PU.1 gene network as a common feature of myeloid leukemia potentially allowing for the identification of patients at risk for malignant transformation. See related commentary by Schleicher and Pietras, p. 378. This article is highlighted in the In This Issue feature, p. 369.
Background: Overactivation of the Transforming growth factor beta (TGF-β) superfamily has been associated with bone marrow failure in MDS. TGF-β binds to set of receptors that include the TGF-β ...receptor I kinase (also known as ALK5), that in turn phosphorylates and activates the downstream SMAD2/3 proteins. Activation of SMAD2/3 transcription factors has been shown to occur in MDS and is associated with anemia. Thus, we wanted to evaluate the preclinical efficacy of novel, clinic-ready, TGF-b receptor I kinase small molecule inhibitors IOA-359 and IOA-360 in MDS models. Results: Using the TREEspot™ SCAN Max Kinase Panel of 403 kinases, IOA-359 and IOA-360 were found to be highly selective for ALK5. Both, IOA-359 and IOA-360 have a blocking activity against ALK5 similar to clinical stage ALK5 inhibitors. To evaluate the efficacy of IOA-359 and IOA-360, we first used HEK 293- Blue-TGF-β cells that stably overexpresses a SMAD inducible secreted embryonic alkaline phosphatase (SEAP) reporter. Both IOA-359 and IOA-360 were able to significantly inhibit TGF-β driven reporter at nanomolar doses ( Fig1A). Next, we showed that both ALK5 inhibitors were able to block TGF-β-mediated SMAD2/3 downstream phosphorylation/activation in a dose dependent manner in MDS and leukemic cell lines. Functionally, the ALK5 inhibitors did not lead to any increase in viability or proliferation in leukemic cell lines. We next tested the efficacy of the novel ALK5 inhibitors in primary patient samples from MDS and AML (N=8). Treatment with both ALK5 inhibitors led to increase in erythroid colony formation in majority of MDS samples in clonogenic assays. Colonies were picked up and examined for erythroid and myeloid differentiation by FACS analysis. We observed that ALK5 inhibition led to increase in Glycophorin A and CD71 expression suggesting enhanced erythroid differentiation ( Fig1B). Myeloid differentiation effects were modest in comparison. Furthermore, in selected samples, addition of Luspatercept led to greater maturation of erythrocytes. Treatment of healthy CD34 + hematopoietic stem and progenitors did not lead to significant changes in differentiation. Lastly, to evaluate downstream effects of ALK5 inhibition, we performed RNAseq analysis in MDS cells. Inhibition with ALK5 inhibitors led to numerous changes in genes affecting cellular differentiation, validating functional changes observed by us. Conclusion: Our current results support the preclinical in vitro efficacy of ALK5 inhibitors IOA-359 and IOA-360 alone and in combination with Luspartercept, highlighting their potential for further development and clinical testing in MDS/AML.
PDF
