Differentiation therapy offers a promising approach in acute myeloid leukemia (AML) by overcoming the developmental block that maintains leukemic blasts. Increasing evidence indicates that DNA replication stress can promote differentiation rather than cytotoxicity; however, the metabolic mechanisms linking replication stress to differentiation remain poorly defined. Here, we investigated how perturbations in nucleotide metabolism regulate replication stress-driven differentiation. Using metabolomic and functional analyses in AML cell lines, we show that agents inducing differentiation through replication stress, including 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr), dihydroorotate dehydrogenase (DHODH) inhibition, and low-dose cytarabine, converge on disruption of nucleotide pool balance. Low-dose AICAr induced a pyrimidine-purine imbalance, S phase arrest, and enhanced differentiation, whereas high-dose reduced these effects. Although brequinar and cytarabine altered nucleotide metabolism through distinct mechanisms, differentiation induced by all agents was abolished by supplementation with high levels of ribo- and deoxyribonucleosides, confirming that nucleotide imbalance is a central driver. We further identify ribonucleotide reductase (RNR) as a critical modulator of this process. Replication stress induced context-dependent regulation of RNR subunits, with RRM2 upregulated in p53-mutant U937 cells and the p53-responsive RRM2B isoform predominating in p53-wild-type MOLM-13 cells. Consistent with these differences, RRM2 depletion enhanced differentiation in U937 cells without affecting viability but impaired differentiation and survival in MOLM-13 cells. These findings position nucleotide metabolism as a key regulator of AML differentiation and suggest that combining RNR-targeted and checkpoint-modulating strategies could optimize therapeutic responses.

How protein quality control is maintained during acute metabolic stress remains poorly understood. In budding yeast, abrupt glucose depletion rapidly lowers ATP levels and leads to the formation of chaperone-containing inclusions, suggesting that ATP-dependent degradation of misfolded proteins may be compromised when energy becomes limiting. Here we find that selective degradation of misfolded proteins remains active during acute glucose starvation despite reduced cellular ATP levels. Using model misfolded substrates in yeast Saccharomyces cerevisiae, we show that misfolded proteins continue to be efficiently degraded throughout both early and late phases of acute glucose depletion. This degradation requires the proteasome and depends on its functional 19S regulatory particle, indicating that ATP-dependent proteasomal activity persists during metabolic stress. We further find that nucleus-vacuole junctions (NVJs) promote efficient degradation during prolonged glucose starvation, revealing a role for organelle contact sites in supporting proteostasis under energy limitation. Together, these findings indicate that cells preserve proteasome-mediated proteostasis during acute glucose starvation, while NVJ membrane contact sites help sustain degradation capacity when metabolic resources are scarce.

Many cells spend a major part of their life in quiescence, a reversible state characterized by a distinct cellular organization and metabolism. In glucose-depleted quiescent yeast cells, there is a metabolic shift from glycolysis to mitochondrial respiration, and a large fraction of proteasomes are reorganized into cytoplasmic granules containing disassembled particles. Given these changes, the operation of protein quality control (PQC) in quiescent cells, in particular the reliance on degradation-mediated PQC and the specific pathways involved, remains unclear. By examining model misfolded proteins expressed in glucose-depleted quiescent yeast cells, we found that misfolded proteins are targeted for selective degradation requiring functional 26S proteasomes. This indicates that a significant pool of proteasomes remains active in degrading quality control substrates. Misfolded proteins were degraded in a manner dependent on the E3 ubiquitin ligases Ubr1 and San1, with Ubr1 playing a dominant role. In contrast to exponentially growing cells, the efficient clearance of certain misfolded proteins additionally required intact nucleus-vacuole junctions (NVJ) and Cue5-independent selective autophagy. Our findings suggest that proteasome activity, autophagy, and NVJ-dependent degradation operate in parallel. Together the data demonstrate that quiescent cells maintain active PQC that relies primarily on selective protein degradation. The necessity of multiple degradation pathways for the removal of misfolded proteins during quiescence underscores the importance of misfolded protein clearance in this cellular state.

There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, "a site to which MCM is bound in G1" might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, a technique referred to as "Chromatin Endogenous Cleavage", we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least 3 orders of magnitude. Published data quantifying the production of ssDNA during S phase showed clear evidence of replication initiation among the most abundant 1600 of these sites, with replication activity decreasing in concert with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5,500 sites. Specifically, these sites (1) appeared in intergenic nucleosome-free regions that were flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. Furthermore, the high resolution of this technique allowed us to demonstrate a strong bias for detecting Mcm double-hexamers downstream rather than upstream of the ACS, which is consistent with the directionality of Mcm loading by Orc that has been observed in vitro. We conclude that, if sites at which Mcm double-hexamers are loaded can function as replication origins, then DNA replication origins are at least 3-fold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than is widely assumed.

Acute myeloid leukemia (AML) is a heterogeneous group of hematological malignancies characterized by differentiation arrest, high relapse rates, and poor survival. The bone marrow (BM) microenvironment is recognized as a critical mediator of drug resistance and a primary site responsible for AML relapse. Our previous study reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) induces AML cell differentiation by inhibiting pyrimidine synthesis and activating Checkpoint kinase 1. Although the protective effect of BM stroma on leukemia cells in response to cytotoxic drugs is well-documented, its effect on AML differentiation remains less explored. In this study, we investigated the impact of stromal cell lines and primary mesenchymal stromal cells (MSCs) on AML cell line differentiation triggered by AICAr and brequinar, a known dihydroorotate dehydrogenase (DHODH) inhibitor. Our findings indicate that the mouse MS-5 stromal cell line, known for its cytoprotective effects, does not inhibit AML cell differentiation induced by pyrimidine synthesis inhibitors. Interestingly, AICAr caused morphological changes and growth arrest in MS-5 stromal cells via an AMP-activated protein kinase (AMPK)-dependent pathway. Human stromal cell lines HS-5 and HS-27, as well as primary MSCs isolated from patient bone marrow, were superior in promoting AML differentiation compared with mouse cells in response to AICAr and brequinar, with the inhibitors not significantly affecting the stromal cells themselves. In conclusion, our study highlights the supportive role of human BM MSCs in enhancing the differentiation effects of pyrimidine synthesis inhibitors on AML cells, suggesting that AML treatment strategies focusing on differentiation rather than cell killing may be successful in clinical settings.NEW & NOTEWORTHY This study is the first to demonstrate that human stromal cell lines and primary mesenchymal stromal cells from patients enhance the in vitro differentiation of acute myeloid leukemia (AML) cells induced by pyrimidine synthesis inhibitors, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr), and brequinar. Furthermore, this is the first report to show that AICAr affects mouse bone marrow stromal cells by activating AMP-activated protein kinase (AMPK) and that human stromal cells are superior to mouse cells for testing the effects of drugs on AML differentiation.

There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, ‘a site that binds Mcm in G1’ might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here, we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least three orders of magnitude. Published data quantifying single-stranded DNA (ssDNA) during S phase revealed replication initiation among the most abundant 1600 of these sites, with replication activity decreasing with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5500 sites. Specifically, these sites: (1) appeared in intergenic nucleosome-free regions flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. We conclude that, if sites at which Mcm double hexamers are loaded can function as replication origins, then DNA replication origins are at least threefold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than widely assumed.

Supplementary Figure 2 from Antitumor Activity of a Small-Molecule Inhibitor of Human Silent Information Regulator 2 Enzymes

Timely completion of genome replication is a prerequisite for mitosis, genome integrity, and cell survival. A challenge to this timely completion comes from the need to replicate the hundreds of untranscribed copies of rDNA that organisms maintain in addition to the copies required for ribosome biogenesis. Replication of these rDNA arrays is relegated to late S phase despite their large size, repetitive nature, and essentiality. Here, we show that, in Saccharomyces cerevisiae, reducing the number of rDNA repeats leads to early rDNA replication, which results in delaying replication elsewhere in the genome. Moreover, cells with early-replicating rDNA arrays and delayed genome-wide replication aberrantly release the mitotic phosphatase Cdc14 from the nucleolus and enter anaphase prematurely. We propose that rDNA copy number determines the replication time of the rDNA locus and that the release of Cdc14 upon completion of rDNA replication is a signal for cell cycle progression.

Supplementary Fig. S2 from Identification and characterization of small-molecule inhibitors of hepsin

Supplementary Fig. S1 from Identification and characterization of small-molecule inhibitors of hepsin

Supplementary Methods from The Androgen-Regulated Protease TMPRSS2 Activates a Proteolytic Cascade Involving Components of the Tumor Microenvironment and Promotes Prostate Cancer Metastasis

Supplementary Figure S1. TMPRSS2 expression is associated with androgen receptor (AR) levels and is regulated by androgens in vivo. Supplementary Figure S2. Characterization of the differentiation status, Tmprss2 and AR expression in Tmprss2+/+ TRAMP and Tmprss2 -/- TRAMP mice. Supplementary Figure S3. Suppression of TMPRSS2 inhibits cell proliferation and invasion. Supplementary Figure S4. TMPRSS2 influences tumor cell clearance and growth in metastatic sites. Supplementary Figure S5. Comparison of cleavage specificities for type II transmembrane serine proteases expressed in prostate epithelium. Supplementary Figure S6. Identification of putative TMPRSS2 substrates based on peptide cleavage specificities determined by PS-SCL. Supplementary Figure S7. Assessment of TMPRSS2 substrate activity. Supplementary Figure S8. Characterization of TMPRSS2 chemical inhibitors. Supplementary Figure S9. Bromhexine suppresses prostate cancer cell invasion but but does not influence proliferation in vitro.

Low-dose cytarabine (LDAC) is a standard therapy for elderly acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. While high doses of cytarabine induce cytotoxicity, the precise mechanism of action of LDAC in AML remains elusive. In vitro studies have demonstrated LDAC-induced differentiation; however, such differentiation is seldom observed in vivo. We hypothesize that this discrepancy may be attributed to the influence of bone marrow (BM) stromal cells on AML cells. Thus, this study aimed to investigate the impact of BM stromal cells on LDAC-induced differentiation of AML cell lines and primary samples. Our results demonstrate that the presence of MS-5 stromal cells prevented LDAC-induced cell cycle arrest, DNA damage signaling and differentiation of U937 and MOLM-13 cell lines. Although transcriptomic analysis revealed that the stroma reduces the expression of genes involved in cytokine signaling and oxidative stress, data obtained with pharmacological inhibitors and neutralizing antibodies did not support the role for CXCL12, TGF-β1 or reactive oxygen species. The presence of stromal cells reduces LDAC-induced differentiation in primary samples from AML-M4 and myelodysplastic syndrome/AML patients. In conclusion, our study demonstrates that BM stroma reduces differentiation of AML induced by LDAC. These findings provide insights into the limited occurrence of terminal differentiation observed in AML patients, and suggest a potential explanation for this observation.

Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors.

All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we mapped the binding sites of the Mcm2-7 helicase complex (MCM) in budding yeast, fission yeast, mouse and humans. We observed similar individual MCM double-hexamer (DH) footprints across the species, but notable differences in their distribution: Footprints in budding yeast were more sharply focused compared to the other three organisms, consistent with the relative sequence specificity of replication origins in S. cerevisiae. Nonetheless, with some clear exceptions, most notably the inactive X-chromosome, much of the fluctuation in replication timing along the chromosomes in all four organisms reflected uneven chromosomal distribution of pre-replication complexes.

Beneficial mutations that arise in an evolving asexual population may compete or interact in ways that alter the overall rate of adaptation through mechanisms such as clonal or functional interference. The application of multiple selective pressures simultaneously may allow for a greater number of adaptive mutations, increasing the opportunities for competition between selectively advantageous alterations, and thereby reducing the rate of adaptation. We evolved a strain of Saccharomyces cerevisiae that could not produce its own histidine or uracil for ~500 generations under one or three selective pressures: limitation of the concentration of glucose, histidine, and/or uracil in the media. The rate of adaptation was obtained by measuring evolved relative fitness using competition assays. Populations evolved under a single selective pressure showed a statistically significant increase in fitness on those pressures relative to the ancestral strain, but the populations evolved on all three pressures did not show a statistically significant increase in fitness over the ancestral strain on any single pressure. Simultaneously limiting three essential nutrients for a population of S. cerevisiae effectively slows the rate of evolution on any one of the three selective pressures applied, relative to the single selective pressure cases. We identify possible mechanisms for fitness changes seen between populations evolved on one or three limiting nutrient pressures by high‐throughput sequencing. Adding multiple selective pressures to evolving disease like cancer and infectious diseases could reduce the rate of adaptation and thereby may slow disease progression, prolong drug efficacy and prevent deaths.

BackgroundAll-trans retinoic acid (ATRA)-based treatment of acute promyelocytic leukemia (APL) is the most successful pharmacological treatment of acute myeloid leukemia (AML). Recent development of inhibitors of mutated isocitrate dehydrogenase and dihydroorotate dehydrogenase (DHODH) has revived interest in differentiation therapy of non-APL AML. Our previous studies demonstrated that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) induced differentiation of monocytic cell lines by activating the ATR/Chk1 via pyrimidine depletion. In the present study, the effects of AICAr on the viability and differentiation of primary AML blasts isolated from bone marrow of patients with non-APL AML were tested and compared with the effects of DHODH inhibitor brequinar and ATRA.MethodsBone marrow samples were obtained from 35 patients and leukemia blasts were cultured ex vivo. The cell viability was assessed by MTT assay and AML cell differentiation was determined by flow cytometry and morphological analyses. RNA sequencing and partial data analysis were conducted using ClusterProfiler package. Statistical analysis was performed using GraphPad Prism 6.0.ResultsAICAr is capable of triggering differentiation in samples of bone marrow blasts cultured ex vivo that were resistant to ATRA. AICAr-induced differentiation correlates with proliferation and sensitivity to DHODH inhibition. RNA-seq data obtained in primary AML blasts confirmed that AICAr treatment induced downregulation of pyrimidine metabolism pathways together with an upregulation of gene set involved in hematopoietic cell lineage.ConclusionAICAr induces differentiation in a subset of primary non-APL AML blasts, and these effects correlate with sensitivity to a well-known, potent DHODH inhibitor.

Genetic ablation as well as pharmacological inhibition of sirtuin 2 (SIRT2), an NAD+-dependent protein deacylase, have therapeutic effects in various cancers and neurodegenerative diseases. Previously, we described the discovery of a dual SIRT1/SIRT2 inhibitor called cambinol (IC50 56 and 59 µM, respectively), which showed cytotoxic activity against cancer cells in vitro and a marked anti-proliferative effect in a Burkitt lymphoma mouse xenograft model. A number of recent studies have shown a protective effect of SIRT1 and SIRT3 in neurodegenerative and metabolic diseases as well as in certain cancers prompting us to initiate a medicinal chemistry effort to develop cambinol-based SIRT2-specific inhibitors devoid of SIRT1 or SIRT3 modulating activity. Here we describe potent cambinol-based SIRT2 inhibitors, several of which show potency of ~600 nM with >300 to >800-fold selectivity over SIRT1 and 3, respectively. In vitro, these inhibitors are found to be toxic to lymphoma and epithelial cancer cell lines. In particular, compounds 55 (IC50 SIRT2 0.25 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) and 56 (IC50 SIRT2 0.78 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) showed apoptotic as well as strong anti-proliferative properties against B-cell lymphoma cells.

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during S-phase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we identified the binding sites of the Mcm2-7 helicase complex, a key component of the pre-RC that is required for initiation of DNA replication, in budding yeast, fission yeast and mouse. In budding yeast, we detected Mcm2 binding in sharply focused peaks, generally with a single double hexamer bound at known origins of replication. In fission yeast, Mcm2 binding, while still concentrated at known origins, was more diffuse, often with 6 to 8 helicase complexes distributed along 0.5-1.5 kb sized origins, and with significantly more binding between origins. Finally, in mouse, we found even more diffuse Mcm2-7 distribution, with the density of Mcm2-7 binding in G1 recapitulating to a remarkable degree the replication program implemented in S-phase. Computer simulations that assign each licensed origin an equal probability of firing show that the observed Mcm2-7 density distribution in G1 across all three species largely recapitulated the DNA replication program. We conclude that the pattern of origin licensing from yeast to mammals is sufficient to explain most differences in replication timing without invoking an overarching temporal program of origin firing.