ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia.

Metabolic reprogramming is a common feature of many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators that promote AML metabolic reprogramming and the benefits conferred to leukemia cells by these metabolic changes remain largely unknown. We report that the transcription factor ATF3 coordinates serine and nucleotide metabolism to maintain cell cycling, survival, and the differentiation blockade in AML. Analysis of mouse and human AML models demonstrate that ATF3 directly activates the transcription of genes encoding key enzymatic regulators of serine synthesis, one-carbon metabolism, and de novo purine and pyrimidine synthesis. Total steady-state polar metabolite and heavy isotope tracing analyses show that ATF3 inhibition reduces de novo serine synthesis, impedes the incorporation of serine-derived carbons into newly synthesized purines, and disrupts pyrimidine metabolism. Importantly, exogenous nucleotide supplementation mitigates the anti-leukemia effects of ATF3 inhibition. Together, these findings reveal the dependence of AML on ATF3-regulated serine and nucleotide metabolism.

Non-Coding RNAs Are Implicit in Chronic Myeloid Leukemia Therapy Resistance.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm initiated by the presence of the fusion gene BCR::ABL1. The development of tyrosine kinase inhibitors (TKIs) highly specific to p210BCR-ABL1, the constitutively active tyrosine kinase encoded by BCR::ABL1, has greatly improved the prognosis for CML patients. Now, the survival rate of CML nearly parallels that of age matched controls. However, therapy resistance remains a persistent problem in the pursuit of a cure. TKI resistance can be attributed to both BCR::ABL1 dependent and independent mechanisms. Recently, the role of non-coding RNAs (ncRNAs) has been increasingly explored due to their frequent dysregulation in a variety of malignancies. Specifically, microRNAs (miRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs) have been shown to contribute to the development and progression of therapy resistance in CML. Since each ncRNA exhibits multiple functions and is capable of controlling gene expression, they exert their effect on CML resistance through a diverse set of mechanisms and pathways. In most cases ncRNAs with tumor suppressing functions are silenced in CML, while those with oncogenic properties are overexpressed. Here, we discuss the relevance of many aberrantly expressed ncRNAs and their effect on therapy resistance in CML.

Knockout of both miR-15/16 loci induces acute myeloid leukemia.

MicroRNAs (miRNAs) have been extensively reported to be associated with hematological malignancies. The loss of miR-15a/16-1 at chromosome 13q14 is a hallmark of most of human chronic lymphocytic leukemia (CLL). Deletion of murine miR-15a/16-1 and miR-15b/16-2 has been demonstrated to promote B cell malignancies. Here, we evaluate the biological role of miR-15/16 clusters, crossbreeding miR-15a/16-1 and miR-15b/16-2 knockout mice. Unexpectedly, the complete deletion of both clusters promoted myeloproliferative disorders in the majority of the mice by the age of 5 months with a penetrance of 70%. These mice showed a significant enlargement of spleen and abnormal swelling of lymph nodes. Flow cytometry characterization demonstrated an expanded CD11b/Gr-1 double-positive myeloid population both in spleen and in bone marrow. The transplantation of splenocytes harvested from double-KO mice into wild-type recipient mice resulted in the development of myeloproliferative disorders, as observed in the donors. In vivo, miR-15/16 cluster deletion up-regulated the expression of Cyclin D1, Cyclin D2, and Bcl-2. Taken together, our findings identify a driver oncogenic role for miR-15/16 cluster deletion in different leukocytic cell lineages.

Mutations associated with a 17-gene leukemia stem cell score and the score's prognostic relevance in the context of the European LeukemiaNet classification of acute myeloid leukemia.

Leukemia stem cells (LSC) are more resistant to standard chemotherapy and their persistence during remission can cause relapse, which is still one of the major clinical challenges in the treatment of acute myeloid leukemia (AML). A better understanding of the mutational patterns and the prognostic impact of molecular markers associated with stemness could lead to better clinical management and improve patients' outcomes. We applied a previously described 17-gene expression score comprising genes differently expressed between LSC and leukemic bulk blasts, for 934 adult patients with de novo AML, and studied associations of the 17-gene LSC score with clinical data and mutation status of 81 genes recurrently mutated in cancer and leukemia. We found that patients with a high 17-gene score were older and had more mutations. The 17-gene score was found to have a prognostic impact in both younger (aged <60 years) and older (aged ≥60 years) patients with AML. We also analyzed the 17-gene LSC score in the context of the 2017 European LeukemiaNet genetic-risk classification and found that for younger patients the score refined the classification, and identified patients currently classified in the European LeukemiaNet Favorable-risk category who had a worse outcome.

Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia.

Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis. Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated. Here, we report that levels of both EGFL7 mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells. High EGFL7 mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age <60 y) patients with cytogenetically normal AML. We further show that AML blasts secrete EGFL7 protein and that higher levels of EGFL7 protein are found in the sera from AML patients than in sera from healthy controls. Treatment of patient AML blasts with recombinant EGFL7 in vitro leads to increases in leukemic blast cell growth and levels of phosphorylated AKT. EGFL7 blockade with an anti-EGFL7 antibody reduced the growth potential and viability of AML cells. Our findings demonstrate that increased EGFL7 expression and secretion is an autocrine mechanism supporting growth of leukemic blasts in patients with AML.

The interleukin-3 receptor CD123 targeted SL-401 mediates potent cytotoxic activity against CD34+CD123+ cells from acute myeloid leukemia/myelodysplastic syndrome patients and healthy donors.

Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123- lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a "bridge-to-transplant" before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.

The Rac GTPase effector p21-activated kinase is essential for hematopoietic stem/progenitor cell migration and engraftment.

The p21-activated kinases (Paks) are serine/threonine kinases that are major effectors of the Rho guanosine 5'\x{2011}triphosphatase, Rac, and Cdc42. Rac and Cdc42 are known regulators of hematopoietic stem and progenitor cell (HSPC) function, however, a direct role for Paks in HSPCs has yet to be elucidated. Lin(-)Sca1(+)c-kit(+) (LSK) cells from wild-type mice were transduced with retrovirus expressing Pak inhibitory domain (PID), a well-characterized inhibitor of Pak activation. Defects in marrow homing and in vitro cell migration, assembly of the actin cytoskeleton, proliferation, and survival were associated with engraftment failure of PID-LSK. The PID-LSK demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK), whereas constitutive activation of ERK in these cells led to rescue of hematopoietic progenitor cell proliferation in vitro and partial rescue of Pak-deficient HSPC homing and engraftment in vivo. Using conditional knock-out mice, we demonstrate that among group A Paks, Pak2(-/-) HSPC show reduced homing to the bone marrow and altered cell shape similar to PID-LSK cells in vitro and are completely defective in HSPC engraftment. These data demonstrate that Pak proteins are key components of multiple engraftment-associated HSPC functions and play a direct role in activation of ERK in HSPCs, and that Pak2 is specifically essential for HSPC engraftment.

Lenalidomide-mediated enhanced translation of C/EBPα-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.

Eradicating acute myeloid leukemia in a Mll(PTD/wt):Flt3(ITD/wt) murine model: a path to novel therapeutic approaches for human disease.

The coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll(PTD/wt) and Flt3(ITD/wt) mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll(PTD/wt):Flt3(ITD/wt) AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease.

Rac signaling in osteoblastic cells is required for normal bone development but is dispensable for hematopoietic development.

Hematopoietic stem cells (HSCs) interact with osteoblastic, stromal, and vascular components of the BM hematopoietic microenvironment (HM) that are required for the maintenance of long-term self-renewal in vivo. Osteoblasts have been reported to be a critical cell type making up the HSC niche in vivo. Rac1 GTPase has been implicated in adhesion, spreading, and differentiation of osteoblast cell lines and is critical for HSC engraftment and retention. Recent data suggest a differential role of GTPases in endosteal/osteoblastic versus perivascular niche function. However, whether Rac signaling pathways are also necessary in the cell-extrinsic control of HSC function within the HM has not been examined. In the present study, genetic and inducible models of Rac deletion were used to demonstrate that Rac depletion causes impaired proliferation and induction of apoptosis in the OP9 cell line and in primary BM stromal cells. Deletion of Rac proteins caused reduced trabecular and cortical long bone growth in vivo. Surprisingly, HSC function and maintenance of hematopoiesis in vivo was preserved despite these substantial cell-extrinsic changes. These data have implications for therapeutic strategies to target Rac signaling in HSC mobilization and in the treatment of leukemia and provide clarification to our evolving concepts of HSC-HM interactions.

Mll partial tandem duplication and Flt3 internal tandem duplication in a double knock-in mouse recapitulates features of counterpart human acute myeloid leukemias.

The MLL-partial tandem duplication (PTD) associates with high-risk cytogenetically normal acute myeloid leukemia (AML). Concurrent presence of FLT3-internal tandem duplication (ITD) is observed in 25% of patients with MLL-PTD AML. However, mice expressing either Mll-PTD or Flt3-ITD do not develop AML, suggesting that 2 mutations are necessary for the AML phenotype. Thus, we generated a mouse expressing both Mll-PTD and Flt3-ITD. Mll(PTD/WT):Flt3(ITD/WT) mice developed acute leukemia with 100% penetrance, at a median of 49 weeks. As in human MLL-PTD and/or the FLT3-ITD AML, mouse blasts exhibited normal cytogenetics, decreased Mll-WT-to-Mll-PTD ratio, loss of the Flt3-WT allele, and increased total Flt3. Highlighting the adverse impact of FLT3-ITD dosage on patient survival, mice with homozygous Flt3-ITD alleles, Mll(PTD/WT):Flt3(ITD/ITD), demonstrated a nearly 30-week reduction in latency to overt AML. Here we demonstrate, for the first time, that Mll-PTD contributes to leukemogenesis as a gain-of-function mutation and describe a novel murine model closely recapitulating human AML.

Rac2-MRC-cIII-generated ROS cause genomic instability in chronic myeloid leukemia stem cells and primitive progenitors.

Chronic myeloid leukemia in chronic phase (CML-CP) is induced by BCR-ABL1 oncogenic tyrosine kinase. Tyrosine kinase inhibitors eliminate the bulk of CML-CP cells, but fail to eradicate leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) displaying innate and acquired resistance, respectively. These cells may accumulate genomic instability, leading to disease relapse and/or malignant progression to a fatal blast phase. In the present study, we show that Rac2 GTPase alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III (MRC-cIII), thereby generating high levels of reactive oxygen species (ROS) in CML-CP LSCs and primitive LPCs. MRC-cIII-generated ROS promote oxidative DNA damage to trigger genomic instability, resulting in an accumulation of chromosomal aberrations and tyrosine kinase inhibitor-resistant BCR-ABL1 mutants. JAK2(V617F) and FLT3(ITD)-positive polycythemia vera cells and acute myeloid leukemia cells also produce ROS via MRC-cIII. In the present study, inhibition of Rac2 by genetic deletion or a small-molecule inhibitor and down-regulation of mitochondrial ROS by disruption of MRC-cIII, expression of mitochondria-targeted catalase, or addition of ROS-scavenging mitochondria-targeted peptide aptamer reduced genomic instability. We postulate that the Rac2-MRC-cIII pathway triggers ROS-mediated genomic instability in LSCs and primitive LPCs, which could be targeted to prevent the relapse and malignant progression of CML.

Guanine nucleotide exchange factor Vav1 regulates perivascular homing and bone marrow retention of hematopoietic stem and progenitor cells.

Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depend on their ability to respond to extracellular signals from the bone marrow microenvironment, but the critical intracellular pathways integrating these signals remain poorly understood. Furthermore, recent studies provide contradictory evidence of the roles of vascular versus osteoblastic niche components in HSPC function. To address these questions and to dissect the complex upstream regulation of Rac GTPase activity in HSPC, we investigated the role of the hematopoietic-specific guanine nucleotide exchange factor Vav1 in HSPC localization and engraftment. Using intravital microscopy assays, we demonstrated that transplanted Vav1(-/-) HSPC showed impaired early localization near nestin(+) perivascular mesenchymal stem cells; only 6.25% of Vav1(-/-) HSPC versus 45.8% of wild-type HSPC were located less than 30 µm from a nestin(+) cell. Abnormal perivascular localization correlated with decreased retention of Vav1(-/-) HSPC in the bone marrow (44-60% reduction at 48 h posttransplant, compared with wild-type) and a very significant defect in short- and long-term engraftment in competitive and noncompetitive repopulation assays (<1.5% chimerism of Vav1(-/-) cells vs. 53-63% for wild-type cells). The engraftment defect of Vav1(-/-) HSPC was not related to alterations in proliferation, survival, or integrin-mediated adhesion. However, Vav1(-/-) HSPC showed impaired responses to SDF1α, including reduced in vitro migration in time-lapse microscopy assays, decreased circadian and pharmacologically induced mobilization in vivo, and dysregulated Rac/Cdc42 activation. These data suggest that Vav1 activity is required specifically for SDF1α-dependent perivascular homing of HSPC and suggest a critical role for this localization in retention and subsequent engraftment.

circPCMTD1 : A protein-coding circular RNA that regulates DNA damage response in BCR/ABL -positive leukemias.

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD 1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.

Epidermal growth factor-like 7 is a novel therapeutic target in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive, noncurative, mature B-cell lymphoma, with a median overall survival of 6-7 years. This underlines a need for effective therapeutic strategies to treat MCL better. Epidermal growth factor-like 7 (EGFL7) is a protein secreted by endothelial cells shown to play a critical role in angiogenesis. Our laboratory has previously demonstrated that EGFL7 supports the growth of leukemic blasts in patients with acute myeloid leukemia (AML); however, its role in MCL has not been investigated yet. In this study, we report that EGFL7 messenger RNA (mRNA) is increased in the cells of patients with MCL compared with cells from healthy controls, and patients with high EGFL7 are associated with lower overall survival rates. Furthermore, EGFL7 is increased in the plasma of patients with MCL compared with the plasma from healthy controls. We further show that EGFL7 binds to epidermal growth factor receptor (EGFR) and activates AKT signaling pathway in MCL cells and that blocking EGFL7 in MCL in patient and cell lines decreases cell growth and increases apoptosis in vitro. Finally, anti-EGFL7 treatment inhibits tumor size and prolongs survival in a mouse model of MCL. In conclusion, our study reveals a role for EGFL7 in MCL cell proliferation and highlights EGFL7 inhibition as a promising new treatment for patients with MCL.

In vivo role of Flt3 ligand and dendritic cells in NK cell homeostasis.

IL-15 is required for NK cell development and homeostasis in vivo. Because IL-15 is presented in trans via its high-affinity IL-15Ralpha-chain to cells expressing the IL-15Rbetagamma complex, we postulated that certain IL-15-bearing cells must be required for NK cell homeostasis. Using IL-15(WT/WT) and IL-15(-/-) mice, bone marrow chimeras with normal cellularity, and a selective depletion of CD11c(hi) dendritic cells (DCs), we demonstrate that ablation of the resting CD11c(hi) DC population results in a highly significant decrease in the absolute number of mature NK cells. In contrast, administration of Flt3 ligand increases the CD11c(hi) DC population, which, when expressing IL-15, significantly expands mature NK cells via enhanced survival and proliferation. In summary, a CD11c(hi) DC population expressing IL-15 is required to maintain NK cell homeostasis under conditions of normal cellularity and also is required to mediate Flt3 ligand-induced NK cell expansion in vivo.

Corrigendum: Role of endothelial cells in graft-versus-host disease.

[This corrects the article DOI: 10.3389/fimmu.2022.1033490.].