ULK2 Is a Key Pro-Autophagy Protein That Contributes to the High Chemoresistance and Disease Relapse in FLT3-Mutated Acute Myeloid Leukemia.

We recently demonstrated that a small subset of cells in FLT3-mutated acute myeloid leukemia (AML) cell lines exhibit SORE6 reporter activity and cancer stem-like features including chemoresistance. To study why SORE6+ cells are more chemoresistant than SORE6- cells, we hypothesized that these cells carry higher autophagy, a mechanism linked to chemoresistance. We found that cytarabine (Ara-C) induced a substantially higher protein level of LC3B-II in SORE6+ compared to SORE6- cells. Similar observations were made using a fluorescence signal-based autophagy assay. Furthermore, chloroquine (an autophagy inhibitor) sensitized SORE6+ but not SORE6- cells to Ara-C. To decipher the molecular mechanisms underlying the high autophagic flux in SORE6+ cells, we employed an autophagy oligonucleotide array comparing gene expression between SORE6+ and SORE6- cells before and after Ara-C treatment. ULK2 was the most differentially expressed gene between the two cell subsets. To demonstrate the role of ULK2 in conferring higher chemoresistance in SORE6+ cells, we treated the two cell subsets with a ULK1/2 inhibitor, MRT68921. MRT68921 significantly sensitized SORE6+ but not SORE6- cells to Ara-C. Using our in vitro model for AML relapse, we found that regenerated AML cells contained higher ULK2 expression compared to pretreated cells. Importantly, inhibition of ULK2 using MRT68921 prevented in vitro AML relapse. Lastly, using pretreatment and relapsed AML patient bone marrow samples, we found that ULK2 expression was higher in relapsed AML. To conclude, our results supported the importance of autophagy in the relapse of FLT3-mutated AML and highlighted ULK2 in this context.

Nuclear NPM-ALK Protects Myc from Proteasomal Degradation and Contributes to Its High Expression in Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma.

In ALK-positive anaplastic large cell lymphoma (ALK+ALCL), a small subset of cancer stem-like (or RR) cells characterized by high Myc expression have been identified. We hypothesize that NPM-ALK contributes to their high Myc expression. While transfection of NPM-ALK into HEK293 cells effectively increased Myc by inhibiting its proteosomal degradation (PD-Myc), this effect was dramatically attenuated when the full-length NPM1 (FL-NPM1) was downregulated using shRNA, highlighting the importance of the NPM-ALK:FL-ALK heterodimers in this context. Consistent with this concept, immunoprecipitation experiments showed that the heterodimers are abundant only in RR cells, in which the half-life of Myc is substantially longer than the bulk cells. Fbw7γ, a key player in PD-Myc, is sequestered by the heterodimers in RR cells, and this finding correlates with a Myc phosphorylation pattern indicative of ineffective PD-Myc. Using confocal microscopy and immunofluorescence staining, we found that the fusion signal between ALK and FL-NPM1, characteristic of the heterodimers, correlates with the Myc level in ALK+ALCL cells from cell lines and patient samples. To conclude, our findings have revealed a novel oncogenic function of NPM-ALK in the nucleus. Specifically, the NPM-ALK:FL-NPM1 heterodimers increase cancer stemness by blocking PD-Myc and promoting Myc accumulation in the cancer stem-like cell subset.

An In Vitro Model for Acute Myeloid Leukemia Relapse Using the SORE6 Reporter.

Many patients diagnosed with acute myeloid leukemia (AML) relapse within two years of the initial remission. The biology of AML relapse is incompletely understood, although cancer stem-like (CSL) cells have been hypothesized to be important. To test this hypothesis, we employed SORE6, a reporter designed to detect the transcriptional activity of the embryonic stem cell proteins Oct4 and Sox2, to identify/purify CSL cells in two FLT3-mutated AML cell lines. Both cell lines contained ~10% of SORE6+ cells in the steady state. Compared to SORE6- cells, SORE6+ cells exhibited more characteristics of CSL cells, with significantly higher chemoresistance and rates of spheroid formation. SORE6+ cells had substantially higher expression of Myc and FLT3 proteins, which are drivers of SORE6 activity. Using a mixture of SORE6-/SORE6+ cells that were molecularly barcoded, we generated an in vitro study model for AML relapse. Specifically, after 'in vitro remission' induced by Ara-C, both cell lines regenerated after 13 ± 3 days. Barcode analysis revealed that most of the regenerated cells were derived from the original SORE6+ cells. Regenerated cells exhibited more CSL features than did the original SORE6+ cells, even though a proportion of them lost SORE6 activity. In bone marrow samples from a patient cohort, we found that relapsed blasts expressed significantly higher levels of Myc, a surrogate marker of SORE6 activity, compared to pre-treatment blasts. To conclude, using our in vitro model, we have provided evidence that CSL cells contribute to AML relapse.

Correction: Li et al. Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter. Curr. Issues Mol. Biol. 2021, 43, 543-557.

In the original publication [...].

Identification and Characterization of Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma Using the SORE6 Reporter.

Transcription factors Sox2 and Oct4 are essential in maintaining the pluripotency of embryonic stem cells and conferring stemness in cancer stem-like (CSL) cells. SORE6, an in-vitro reporter system, was designed to quantify the transcription activity of Sox2/Oct4 and identify CSL cells in non-hematologic cancers. Using SORE6, we identified and enriched CSL cells in ALK-positive anaplastic large cell lymphoma (ALK + ALCL). Two ALK + ALCL cell lines, SupM2 and UCONN-L2, contained approximately 20% of SORE6+ cells, which were purified based on their expression of green fluorescent protein. We then performed functional studies using single-cell clones derived from SORE6- and SORE6+ cells. Compared to SORE6- cells, SORE6+ cells were significantly more chemoresistant and clonogenic in colony-formation assays. Sox2/Oct4 are directly involved in conferring these CSL properties, since the shRNA knockdown of Sox2 in SORE6+ significantly lowered their chemoresistance, while enforced expression of Sox2/Oct4 in SORE6- cells produced opposite effects. Using Western blots, we found that the expression and subcellular localization of Sox2/Oct4 were similar between SORE6- and SORE6+ cells. However, in SORE6+ but not SORE6- cells, Sox2 and Oct4 abundantly bound to a probe containing the SORE6 consensus sequence. c-Myc, previously shown to regulate cancer stemness in ALK + ALCL, regulated the SORE6 activity. In conclusion, SORE6 is useful in identifying/enriching CSL cells in ALK + ALCL.

Gene Methylation and Silencing of WIF1 Is a Frequent Genetic Abnormality in Mantle Cell Lymphoma.

We have previously shown that the Wnt canonical pathway (WCP) is constitutively active in most cases of mantle cell lymphoma (MCL). Here, we aimed to elucidate the mechanisms underlying this biochemical deregulation. We hypothesized that gene methylation/silencing of WIF1 (Wnt inhibitory factor-1), a physiologic inhibitor of WCP, contributes to the deregulation of WCP and promotes cell growth in MCL. In support of this hypothesis, we found that the expression of WIF1 was detectable in none of the 4 MCL cell lines, and in only 2 of 5 tumors (40%) examined. Using methylation-specific PCR, we found evidence of gene methylation of WIF1 in 4 of 5 cell lines (80%) and in 24 of 29 (82%) tumors. The addition of the demethylation agent 5-aza-2'-deoxycytidine to Mino and JeKo-1, two WIF1-negative cell lines, restored the expression of WIF1 mRNA in these cells. Gene transfection of WIF1 into JeKo-1 and Mino cells significantly reduced cell growth, and this finding correlated with substantial downregulations of various proteins in WCP, such as ß-catenin and pGSK-3ß. In conclusion, our results support the concept that gene methylation/silencing of WIF1 is a frequent event in MCL, and this abnormality contributes to the aberrant activation of WCP. These results have provided further evidence that aberrant Wnt signaling is pathogenetically important in MCL and it may represent a potential therapeutic target.

Crizotinib Resistance Mediated by Autophagy Is Higher in the Stem-Like Cell Subset in ALK-Positive Anaplastic Large Cell Lymphoma, and This Effect Is MYC-Dependent.

Previously it was shown that autophagy contributes to crizotinib resistance in ALK-positive anaplastic large cell lymphoma (ALK + ALCL). We asked if autophagy is equally important in two distinct subsets of ALK + ALCL, namely Reporter Unresponsive (RU) and Reporter Responsive (RR), of which RR cells display stem-like properties. Autophagic flux was assessed with a fluorescence tagged LC3 reporter and immunoblots to detect endogenous LC3 alongside chloroquine, an autophagy inhibitor. The stem-like RR cells displayed significantly higher autophagic response upon crizotinib treatment. Their exaggerated autophagic response is cytoprotective against crizotinib, as inhibition of autophagy using chloroquine or shRNA against BECN1 or ATG7 led to a decrease in their viability. In contrast, autophagy inhibition in RU resulted in minimal changes. Since the differential protein expression of MYC is a regulator of the RU/RR dichotomy and is higher in RR cells, we asked if MYC regulates the autophagy-mediated cytoprotective effect. Inhibition of MYC in RR cells using shRNA significantly blunted crizotinib-induced autophagic response and effectively suppressed this cytoprotective effect. In conclusion, stem-like RR cells respond with rapid and intense autophagic flux which manifests with crizotinib resistance. For the first time, we have highlighted the direct role of MYC in regulating autophagy and its associated chemoresistance phenotype in ALK + ALCL stem-like cells.

NPM-ALK Is a Key Regulator of the Oncoprotein FOXM1 in ALK-Positive Anaplastic Large Cell Lymphoma.

Forkhead Box M1 (FOXM1) is an oncogenic transcription factor implicated in the pathogenesis of solid and hematologic cancers. In this study, we examined the significance of FOXM1 in NPM-ALK-positive anaplastic large cell lymphoma (NPM-ALK + ALCL), with a focus on how it interacts with NPM-ALK, which is a key oncogenic driver in these tumors. FOXM1 was expressed in NPM-ALK + ALCL cell lines (5/5), patient samples (21/21), and tumors arising in NPM-ALK transgenic mice (4/4). FOXM1 was localized in the nuclei and confirmed to be transcriptionally active. Inhibition of FOXM1 in two NPM-ALK + ALCL cells using shRNA and pharmalogic agent (thiostrepton) resulted in reductions in cell growth and soft-agar colony formation, which were associated with apoptosis and cell-cycle arrest. FOXM1 is functionally linked to NPM-ALK, as FOXM1 enhanced phosphorylation of the NPM-ALK/STAT3 axis. Conversely, DNA binding and transcriptional activity of FOXM1 was dependent on the expression of NPM-ALK. Further studies showed that this dependency hinges on the binding of FOXM1 to NPM1 that heterodimerizes with NPM-ALK, and the phosphorylation status of NPM-ALK. In conclusion, we identified FOXM1 as an important oncogenic protein in NPM-ALK+ ALCL. Our results exemplified that NPM-ALK exerts oncogenic effects in the nuclei and illustrated a novel role of NPM1 in NPM-ALK pathobiology.

Xanthogranulomatous prostatitis presenting as Pseudomonas aeruginosa prostatic abscesses: An uncommon complication after kidney transplantation.

Xanthogranulomatous (XG) prostatitis is a rare form of granulomatous prostatitis characterized by a benign inflammatory process of non-specific etiology that clinically may mimic carcinoma. Few cases have been reported in the English language medical literature, with only four reported cases presenting as prostatic abscesses. A 70-year-old male with type 2 diabetes mellitus and two previous kidney transplants presented with septic shock secondary to Pseudomonas aeruginosa bacteremia 4 days after undergoing a cystoscopy. Despite appropriate antimicrobial therapy, P. aeruginosa persisted in the blood for a total of 7 days. There were no indwelling prosthetic devices, no complicated pyelonephritis, and no endovascular sources of infection. Upon repeat clinical assessment, the patient reported pelvic pain. A digital rectal examination revealed prostatic tenderness and an endorectal ultrasound confirmed multiple prostatic abscesses. An ultrasound-guided transrectal needle aspirate drained scant purulent fluid and cultures grew the same phenotypic strain of P. aeruginosa. For definitive source control, the patient underwent transurethral resection of the prostate with unroofing of prostatic abscesses. The pathological findings were diagnostic of XG prostatitis. Given the rather acute presentation of this case, our hypothesis is that the prior urological instrumentation likely facilitated bacterial translocation and created the ideal environment for the development of pseudomonal prostatic abscesses resulting in XG inflammation and necrosis. XG prostatitis is a rare entity of uncertain etiology that can result in prostatic abscesses, and surgery is required for definitive diagnosis and management.

Assessing differential expression in two-color microarrays: a resampling-based empirical Bayes approach.

Microarrays are widely used for examining differential gene expression, identifying single nucleotide polymorphisms, and detecting methylation loci. Multiple testing methods in microarray data analysis aim at controlling both Type I and Type II error rates; however, real microarray data do not always fit their distribution assumptions. Smyth's ubiquitous parametric method, for example, inadequately accommodates violations of normality assumptions, resulting in inflated Type I error rates. The Significance Analysis of Microarrays, another widely used microarray data analysis method, is based on a permutation test and is robust to non-normally distributed data; however, the Significance Analysis of Microarrays method fold change criteria are problematic, and can critically alter the conclusion of a study, as a result of compositional changes of the control data set in the analysis. We propose a novel approach, combining resampling with empirical Bayes methods: the Resampling-based empirical Bayes Methods. This approach not only reduces false discovery rates for non-normally distributed microarray data, but it is also impervious to fold change threshold since no control data set selection is needed. Through simulation studies, sensitivities, specificities, total rejections, and false discovery rates are compared across the Smyth's parametric method, the Significance Analysis of Microarrays, and the Resampling-based empirical Bayes Methods. Differences in false discovery rates controls between each approach are illustrated through a preterm delivery methylation study. The results show that the Resampling-based empirical Bayes Methods offer significantly higher specificity and lower false discovery rates compared to Smyth's parametric method when data are not normally distributed. The Resampling-based empirical Bayes Methods also offers higher statistical power than the Significance Analysis of Microarrays method when the proportion of significantly differentially expressed genes is large for both normally and non-normally distributed data. Finally, the Resampling-based empirical Bayes Methods are generalizable to next generation sequencing RNA-seq data analysis.

Heat avoidance is regulated by transient receptor potential (TRP) channels and a neuropeptide signaling pathway in Caenorhabditis elegans.

The ability to avoid noxious extremes of hot and cold is critical for survival and depends on thermal nociception. The TRPV subset of transient receptor potential (TRP) channels is heat activated and proposed to be responsible for heat detection in vertebrates and fruit flies. To gain insight into the genetic and neural basis of thermal nociception, we developed assays that quantify noxious heat avoidance in the nematode Caenorhabditis elegans and used them to investigate the genetic basis of this behavior. First, we screened mutants for 18 TRP channel genes (including all TRPV orthologs) and found only minor defects in heat avoidance in single and selected double and triple mutants, indicating that other genes are involved. Next, we compared two wild isolates of C. elegans that diverge in their threshold for heat avoidance and linked this phenotypic variation to a polymorphism in the neuropeptide receptor gene npr-1. Further analysis revealed that loss of either the NPR-1 receptor or its ligand, FLP-21, increases the threshold for heat avoidance. Cell-specific rescue of npr-1 implicates the interneuron RMG in the circuit regulating heat avoidance. This neuropeptide signaling pathway operates independently of the TRPV genes, osm-9 and ocr-2, since mutants lacking npr-1 and both TRPV channels had more severe defects in heat avoidance than mutants lacking only npr-1 or both osm-9 and ocr-2. Our results show that TRPV channels and the FLP-21/NPR-1 neuropeptide signaling pathway determine the threshold for heat avoidance in C. elegans.

The DAF-2 insulin-like signaling pathway independently regulates aging and immunity in C. elegans.

The Caenorhabditis elegans DAF-2 insulin-like signaling pathway, which regulates lifespan and stress resistance, has also been implicated in resistance to bacterial pathogens. Loss-of-function daf-2 and age-1 mutants have increased lifespans and are resistant to a variety of bacterial pathogens. This raises the possibility that the increased longevity and the pathogen resistance of insulin-like signaling pathway mutants are reflections of the same underlying mechanism. Here we report that regulation of lifespan and resistance to the bacterial pathogen Pseudomonas aeruginosa is mediated by both shared and genetically distinguishable mechanisms. We find that loss of germline proliferation enhances pathogen resistance and this effect requires daf-16, similar to the regulation of lifespan. In contrast, the regulation of pathogen resistance and lifespan is decoupled within the DAF-2 pathway. Long-lived mutants of genes downstream of daf-2, such as pdk-1 and sgk-1, show wildtype resistance to pathogens. However, mutants of akt-1 and akt-2, which we find to individually have modest effects on lifespan, show enhanced resistance to pathogens. We also demonstrate that pathogen resistance of daf-2, akt-1, and akt-2 mutants is associated with restricted bacterial colonization, and that daf-2 mutants are better able to clear an infection after challenge with P. aeruginosa. Moreover, we find that pathogen resistance among insulin-like signaling mutants is associated with increased expression of immunity genes during infection. Other processes that affect organismal longevity, including Jun kinase signaling and caloric restriction, do not affect resistance to bacterial pathogens, further establishing that aging and innate immunity are regulated by genetically distinct mechanisms.

Transcriptional regulation and binding of heat shock factor 1 and heat shock factor 2 to 32 human heat shock genes during thermal stress and differentiation.

Transcription of mammalian heat shock genes can be regulated by heat shock factors (HSF) 1 and 2. Although it has been shown previously that these factors respond to distinct stimuli, a broad analysis of the induction and function of these factors in living cells has not been performed. In our study, we assayed binding of human HSF1 and HSF2 at the promoters of 32 genes identified through LocusLink as heat shock genes in response to elevated temperature and hemin-induced differentiation in human K562 erythroleukemic cells using the chromatin immunoprecipitation technique. We also measured the induced expression of these genes under these 2 conditions. We found that 17 of the 32 genes were transcriptionally induced during heat shock, and HSF1 binding was detected at 15 of the 17 promoters. Nearly all the genes induced by heat shock were also induced to a lesser degree during hemin treatment. However, some genes were induced significantly more during hemin treatment than during heat shock. A new finding is that HSF1 and HSF2 bind to the same targets, but HSF1 binding is activated more by heat than by hemin treatment, and HSF2 binding is only activated by hemin treatment and not by heat. This technology also identified previously unknown HSF1 binding sites near genes that were previously shown to be heat inducible that may contribute to gene-specific regulation.

Characterization of mediators of microbial virulence and innate immunity using the Caenorhabditis elegans host-pathogen model.

The soil-borne nematode, Caenorhabditis elegans, is emerging as a versatile model in which to study host-pathogen interactions. The worm model has shown to be particularly effective in elucidating both microbial and animal genes involved in toxin-mediated killing. In addition, recent work on worm infection by a variety of bacterial pathogens has shown that a number of virulence regulatory genes mediate worm susceptibility. Many of these regulatory genes, including the PhoP/Q two-component regulators in Salmonella and LasR in Pseudomonas aeruginosa, have also been implicated in mammalian models suggesting that findings in the worm model will be relevant to other systems. In keeping with this concept, experiments aimed at identifying host innate immunity genes have also implicated pathways that have been suggested to play a role in plants and animals, such as the p38 MAP kinase pathway. Despite rapid forward progress using this model, much work remains to be done including the design of more sensitive methods to find effector molecules and further characterization of the exact interaction between invading pathogens and C. elegans' cellular components.