Pim-1 Proto-oncogene Research Articles

Regulation of Erythroid Differentiation via the HIF1α-NFIL3-PIM1 Signaling Axis Under Hypoxia.

Aims: Erythropoiesis is controlled by several factors, including oxygen level under different circumstances. However, the role of hypoxia in erythroid differentiation and the underlying mechanisms are poorly understood. We studied the effect and mechanism of hypoxia on erythroid differentiation of K562 cells and observed the effect of hypoxia on early erythropoiesis of zebrafish. Results: Compared with normal oxygen culture, both hemin-induced erythroid differentiation of K562 cells and the early erythropoiesis of zebrafish were inhibited under hypoxic treatment conditions. Hypoxia-inducible factor 1 alpha (HIF1α) plays a major role in the response to hypoxia. Here, we obtained a stable HIF1α knockout K562 cell line using the CRISPR-Cas9 technology and further demonstrated that HIF1α knockout promoted hemin-induced erythroid differentiation of K562 cells under hypoxia. We demonstrated an HIF1-mediated induction of the nuclear factor interleukin-3 (NFIL3) regulated in K562 cells under hypoxia. Interestingly, a gradual decrease in NFIL3 expression was detected during erythroid differentiation of erythropoietin-induced CD34+ hematopoietic stem/progenitor cells (HSPCs) and hemin-induced K562 cells. Notably, erythroid differentiation was inhibited by enforced expression of NFIL3 under normoxia and was promoted by the knockdown of NFIL3 under hypoxia in hemin-treated K562 cells. In addition, a target of NFIL3, pim-1 proto-oncogene, serine/threonine kinase (PIM1), was obtained by RNA microarray after NFIL3 knockdown. PIM1 can rescue the inhibitory effect of NFIL3 on hemin-induced erythroid differentiation of K562 cells. Innovation and Conclusion: Our findings demonstrate that the HIF1α-NFIL3-PIM1 signaling axis plays an important role in erythroid differentiation under hypoxia. These results will provide useful clues for preventing the damage of acute hypoxia to erythropoiesis.

Kinase PIM1 governs ferroptosis to reduce retinal microvascular endothelial cell dysfunction triggered by high glucose.

Previous studies have implicated targeting Pim-1 proto-oncogene, serine/threonine kinase (PIM1) as a preventive measure against high glucose-induced cellular stress and apoptosis. This study aimed to reveal the potential role and regulatory mechanism of PIM1 in diabetic retinopathy. Human retinal microvascular endothelial cells (hRMECs) underwent high glucose induction, and fluctuations in PIM1 levels were assessed. By overexpressing PIM1, its effects on the levels of inflammatory factors, oxidative stress indicators, migration and tube formation abilities, tight junction protein expression levels, and ferroptosis in hRMECs were identified. Afterwards, hRMECs were treated with the ferroptosis-inducing agent erastin, and the effect of erastin on the above PIM1 regulatory functions was focused on. PIM1 was downregulated upon high glucose, and its overexpression inhibited the inflammatory response, oxidative stress, cell migration, and tube formation potential in hRMECs, whereas elevated tight junction protein levels. Furthermore, PIM1 overexpression reduced intracellular iron ion levels, lipid peroxidation, and levels of proteins actively involved in ferroptosis. Erastin treatment reversed the impacts of PIM1 on hRMECs, suggesting the mediation of ferroptosis in PIM1 regulation. The current study has yielded critical insights into the role of PIM1 in ameliorating high glucose-induced hRMEC dysfunction through the inhibition of ferroptosis.

GATA1 regulates the microRNA‑328‑3p/PIM1axis via circular RNA ITGB1 to promote renal ischemia/reperfusion injury in HK‑2 cells.

Acute kidney injury (AKI) is caused by renal ischemia/reperfusion injury (IRI) during kidney transplantation. The levels of both circular RNAs (circRNAs) and microRNAs (miRNAs/miR) appear to be critical for AKI detection. While several RNA interactions in AKI have been found, the regulatory mechanisms between the molecules remain to be fully elucidated. In the present study, miRNA expression profiling analysis was conducted using an online dataset to identify the differentially expressed miRNAs in rats with IRI. miR-328-3p was also found to be downregulated in human kidney-2 (HK-2) cells subjected to hypoxia/reperfusion (H/R), and its overexpression targeting pim-1 proto-oncogene (PIM1) resulted in an increased viability and a reduced apoptosis, as well as in the decreased expression of inflammatory factors upon H/R exposure. Putative targets and circRNAs of miR-328-3p were identified using publically available databases. The inhibition of circRNA integrin beta 1 (ITGB1; circITGB1) suppressed the inflammatory response induced by H/R by sponging miR-328-3p in HK-2 cells. Furthermore, a sequence of the functional ITGB1 promoter was studied for transcription factor GATA binding protein 1 (GATA1) binding sites. GATA1 binds to the ITGB1 promoter, leading to the expression of circITGB1. On the whole, the findings of the present study revealed a regulatory pathway modulating miR-328-3p in IRI, demonstrating that the GATA1-mediated regulation of circITGB1 enhanced the H/R-induced inflammatory response via the miR-328-3p/PIM1 axis.

KLF2-induced circZKSCAN1 potentiates the tumorigenic properties of clear cell renal cell carcinoma by targeting the miR-1294/PIM1 axis

ABSTRACT Clear cell renal cell carcinoma (ccRCC) is one of the most common and lethal types of urologic cancer. With low survival rates among patients in advanced stages of disease, and increasing rate of morbidity and mortality worldwide, novel therapeutic targets for ccRCC clinical intervention are necessary. In this study, we investigated the functional role of circZKSCAN1 in ccRCC progression. Our results suggested that circZKSCAN1 was abundantly expressed in ccRCC tumor tissues and cells. CircZKSCAN1 knockdown significantly inhibited cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition of renal cell carcinoma (RCC) cells, whereas potentiated Natural Killer (NK) cell-mediated cytotoxicity against RCC cells in vitro and repressed tumor growth in vivo. Furthermore, we identified a novel circZKSCAN1/miR-1294/PIM1 axis was identified in RCC progression, showing that the expression of circZKSCAN1 expression in RCC cells was transcriptionally regulated by Kruppel-like factor 2. The results of our study may provide new insights for ccRCC basic research. Abbreviations: ccRCC: clear cell renal cell carcinoma; ChIP: chromatin immunoprecipitation; circRNA: circular RNA; EDU: 5‐ethynyl‐2’‐deoxyuridine; EMT: epithelial-mesenchymal transition; FBS: fetal bovine serum; FISH: RNA fluorescent in situ hybridization; KLF2: Kruppel-like factor 2; NC: normal control; NK cell: natural killer cell; NOD/SCID: nonobese severe diabetic/severe combined immunodeficiency; PIM1: Pim-1 proto-oncogene, serine/threonine kinase; RCC: renal cell carcinoma; ZKSCAN1: zinc finger with KRAB and SCAN domains 1

Myeloid-Derived Growth Factor Protects Against Pressure Overload–Induced Heart Failure by Preserving Sarco/Endoplasmic Reticulum Ca 2+ -ATPase Expression in Cardiomyocytes

Background: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation’s potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload. Methods: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf -deficient ( Mydgf −/− ), and Mydgf bone marrow–chimeric or bone marrow–conditional transgenic mice with pressure overload–induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography–mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca 2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF. Results: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf −/− mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow–derived inflammatory cells attenuated pressure overload–induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein–coupled receptor agonist–induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca 2+ -ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf −/− mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca 2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf −/− mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload–induced hypertrophy and dysfunction. Pressure-overloaded Mydgf −/− mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival. Conclusions: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload–induced heart failure.

Genetic Activation and Therapeutic Targeting of PIM1 in T-Cell Acute Lymphoblastic Leukemia/Lymphoma

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell acute lymphoblastic lymphoma (T-LBL) are aggressive immature T-cell malignancies that are often characterized by improper T-cell receptor (TCR) recombinations leading to aberrant activation of proto-oncogenes. Current multi-agent combination chemotherapy treatment provides an overall survival rate of ~80% in children and 50% in adults. However, the prognosis of patients with relapsed and refractory T-ALL/T-LBL remains extremely poor and the development of more effective and specific antileukemic drugs is critical. Therefore, we aim to identify new oncogenes, define their mechanism of action and establish strategies to develop targeted, highly specific antileukemic drugs.Here, we used Targeted Locus Amplification (TLA, de Vree et al., Nat Biotechnol., 2014) to identify a novel TCRβ driven t(6;7)(p21;q34) translocation in a human T-LBL patient resulting in aberrant activation of the PIM1 proto-oncogene. PIM1 is a constitutively active serine/threonine kinase involved in cell cycle progression, apoptosis, transcription and drug resistance and is overexpressed in a variety of human cancers. Its expression is controlled by the JAK/STAT signaling pathway. Further characterization of this PIM1 rearranged patient sample revealed cooperative genetic alterations that target known T-ALL/T-LBL onco- and tumor suppressor genes, including NOTCH1, IKZF1, EP300 and CDKN2A.Comparing PIM1 expression between normal T-cell subsets, T-ALL and T-LBL patient samples using qPCR revealed subsets in both T-LBL and T-ALL patients with high PIM1 levels. This PIM1 expression can be both monoallelicaly (due to translocation) or biallelicaly (due to upstream mutations in f.e. the JAK/STAT pathway). Using publically available gene expression datasets, we show that oncogenic PIM1 activation is largely restricted to immature, TLX1, TLX3, NKX2-1 or HOXA positive T-ALL and T-LBL patients, and is mostly not present in TAL1 or LMO2 rearranged tumors. This pattern of PIM1 activation is in line with the preferential occurrence of JAK-STAT pathway alterations in these genetic subtypes of human T-ALL/T-LBL.To study the oncogenic properties of PIM1 in the context of malignant T-cell transformation, we did RNA sequencing and phosphoproteomics on the T-ALL/T-LBL tumor line HSB-2 (cell line with the highest PIM1 expression) and on ex vivo treated leukemic cells harvested from the spleen of T-LBL engrafted NSG mice after PIM1 inhibition with TP-3654 (Foulks et al., Neoplasia, 2014). These data revealed that PIM1 inhibition has broad effects on transcription and phosphorylation substrates involved in cell cycle, translation and apoptosis. For example, we showed that the oncogenic properties of PIM1 are, at least in part, mediated by phosphorylation of GSK3β and subsequent stabilization of anti-apoptotic MCL1.Next, we evaluated the therapeutic potential of PIM1 inhibition. Daily TP-3654 treatment for 4 weeks of T-LBL engrafted NSG mice resulted in strong anti-leukemic effects. In vitro combination treatment with TP-3654 and glucocorticoids showed strong synergism, while the combination with other chemotherapeutics such as asparaginase and vincristine did not. Moreover, the combination of TP-3654 and the glucocorticoid dexamethasone in vivo almost completely cured the leukemic mice (<1% hCD45 positive blasts in the peripheral blood).All together, our study identifies PIM1 as a putative oncogene in T-ALL and T-LBL and suggests that inhibition of this serine/threonine kinase could serve as a novel therapeutic strategy in these aggressive T-cell neoplasms. DisclosuresNo relevant conflicts of interest to declare.

Sialylated Glycans Regulate MUC13 and the Proto-Oncogenes Pim-1 and Myc to Control Hematopoietic Stem and Progenitor Cell Numbers

The enzyme β-1-4 galactosyltransferase 1 (β4GalT1) plays a critical role in thrombopoiesis by modulating sialo-glycan (sialyl N-acetyl-lactosamine or LacNAc) content and function of the β1 integrin on megakaryocytes (MKs) (Nat. Commun. 2020;11(1):356). Recent data, however, point to a more complex role for β4GalT1 in hematopoiesis, as the promoter region of its conserved gene, B4galt1, is rich in enhancer sequences for transcription factors associated with cell identity and pro-oncogenic regulatory programs. Here, we investigated the homeostasis of hematopoietic stem and progenitor cells (HSPCs) in B4galt1-/- mice. We demonstrate that lack of Sialylated LacNAc synthesis perturbs HSPCs beyond the homing deficiency associated with lack of homing sialo-glycan motifs. Flow cytometry analysis showed that B4galt1-/- mouse bone marrows have increased numbers of Long-Term HSCs (LT-HSCs, defined as LineageNeg/Sca-1Pos/c-KitPos/CD150Pos/CD48Neg). The increase in HSC numbers led us to investigate their phenotypic and functional features further. While quiescence markers in B4galt1-/- LT-HSCs were indistinguishable relative to controls, LT-HSCs expressed more of the platelet marker CD41 on their surface, supporting a highly expanded CD41+ subset of LT-HSCs. Platelet-bias of LT-HSCs has been associated with inflammation and aging. However, our data do not support an increased cytokine inflammatory profile in the bone marrow. Instead, single-cell RNA sequencing (scRNA seq) of sorted β4galt1-/- LineageNeg/Sca-1Pos/c-KitPos (LSK) cells showed a significantly increased expression of the proto-oncogene Pim-1, its target, Myc, and the heavily O-glycosylated transmembrane receptor mucin 13 (MUC13), compared to control cells. Analysis of LT-HSC glycan expression using lectin microarray showed the expected decrease in N-glycosylation associated with B4galt1 deficiency, but also an increase in O-glycans, consistent with overexpression of MUC13, expression of which was enriched compared to other surface mucins. The data show that B4galt1 deletion leads to overexpression of the proto-oncogenes Pim-1, Myc, and MUC13 in HSPCs. The data suggest that MUC13-associated O-glycans and glyco-synthetic genes are potential therapeutic targets for hematologic malignancies since mucins have anti-inflammatory functions and alterations in mucin expression are with inflammation and cancer. Disclosures No relevant conflicts of interest to declare.

Arsenic trioxide-induced upregulation of miR-1294 suppresses tumor growth in hepatocellular carcinoma by targeting TEAD1 and PIM1.

Recently, Arsenic trioxide (ATO) has been reported as an efficient drug for suppression of cancer cell growth. Existing studies revealed the extensive involvement of microRNAs (miRNAs) in initiation and development of hepatocellular carcinoma (HCC). However, the potential correlation between ATO and miRNAs in HCC progression remains to be explored. To conduct our research, we applied a qRT-PCR analysis to find miRNAs that were upregulated in HCC cells treated with ATO. In our present study, miR-1294 was found to be significantly upregulated in ATO-treated HCC cells. To confirm the function of ATO and miR-1294 in HCC progression, gain-of function assays were designed and conducted. As expected, proliferative ability of ATO-treated HCC cells was markedly weakened compared to DMSO-treated HCC cells. More importantly, proliferation was further suppressed in ATO-induced HCC cells after overexpression of miR-1294. Through bioinformatics analysis, some potential targets of miR-1294 were predicted. Further investigation revealed that Pim-1 proto-oncogene (PIM1) and TEA domain transcription factor 1 (TEAD1) were two downstream targets of miR-1294 and could be negatively regulated by ATO. Functionally, we determined that cell proliferation and apoptosis resistance suppressed by miR-1294 and ATO were recovered by introduction of TEAD1 and PIM1. Collectively, this study revealed that a novel ATO-miR-1294-TEAD1/PIM1 axis regulated HCC cell growth, offering a potential insight into the HCC therapy.

Downregulation of miRNA‑328 promotes the angiogenesis of HUVECs by regulating the PIM1 and AKT/mTOR signaling pathway under high glucose and low serum condition.

Vascular complications are the primary reason for disability and mortality associated with diabetes mellitus (DM), and numerous microRNAs (miRNAs/miRs) are involved in the process, such as miR-122, miR-24 and miR-423. It has been reported that miR-328 regulates DM and cardiovascular disease; however, the role and mechanism of action underlying miR-328 in HUVECs is not completely understood. The present study aimed to investigate the role and mechanism of action underlying the effects of miR-328 on the functions of HUVECs. To simulate hyperglycemia combined with ischemia-induced tissue starvation, HUVECs were cultured in endothelial cell medium with 25 mmol/l D-glucose and 2% FBS for 24 h [high glucose (HG) + 2% FBS group]. HUVEC miR-328 expression levels were detected by reverse transcription-quantitative PCR. Cell migration, cytotoxicity and tube-like structure formation were analyzed using wound healing, Cell Counting Kit-8 and tube formation assays, respectively. Following transfection with miR-328 inhibitor, miR-328 expression was downregulated in HUVECs. Protein expression levels were determined by western blotting. Compared with the control group, the migration and tube-like structure formation of HUVECs were decreased, and cell cytotoxicity was increased in the HG + 2% FBS group. The protein expression levels of vascular endothelial growth factor were also decreased, and the expression levels of miRNA-328 in the HG + 2% FBS group were increased compared with the control group. However, miRNA-328 downregulation reversed the aforementioned effects. Further experiments indicated that the AKT signaling pathway was inhibited in the HG + 2% FBS group; however, miR-328 downregulation activated the AKT/mTOR signaling pathway, which was blocked by the AKT signaling pathway inhibitor, perifosine. Gene prediction and western blotting demonstrated that miR-328 displayed a regulatory role via Pim-1 proto-oncogene, serine/threonine kinase (PIM1). In conclusion, miR-328 expression was upregulated and angiogenesis was inhibited when HUVECs were subjected to high glucose and low serum conditions. miR-328 downregulation enhanced angiogenesis by increasing PIM1 expression and activating the AKT/mTOR signaling pathway in HUVECs under high glucose and low serum conditions.

The extracellular sulfatase SULF2 promotes liver tumorigenesis by stimulating assembly of a promoter-looping GLI1-STAT3 transcriptional complex

The expression of the extracellular sulfatase SULF2 has been associated with increased hepatocellular carcinoma (HCC) growth and poor patient survival. However, the molecular mechanisms underlying SULF2-associated tumor growth remain unclear. To address this gap, here we developed a transgenic mouse overexpressing Sulf2 in hepatocytes under the control of the transthyretin promoter. In this model, Sulf2 overexpression potentiated diethylnitrosamine-induced HCC. Further analysis indicated that the transcription factor GLI family zinc finger 1 (GLI1) mediates Sulf2 expression during HCC development. A cross of the Sulf2-overexpressing with Gli1-knockout mice revealed that Gli1 inactivation impairs SULF2-induced HCC. Transcriptomic analysis revealed that Sulf2 overexpression is associated with signal transducer and activator of transcription 3 (STAT3)-specific gene signatures. Interestingly, the Gli1 knockout abrogated SULF2-mediated induction of several STAT3 target genes, including suppressor of cytokine signaling 2/3 (Socs2/3); Pim-1 proto-oncogene, Ser/Thr kinase (Pim1); and Fms-related tyrosine kinase 4 (Flt4). Human orthologs were similarly regulated by SULF2, dependent on intact GLI1 and STAT3 functions in HCC cells. SULF2 overexpression promoted a GLI1-STAT3 interaction and increased GLI1 and STAT3 enrichment at the promoters of their target genes. Interestingly, the SULF2 overexpression resulted in GLI1 enrichment at select STAT3 consensus sites, and vice versa. siRNA-mediated STAT3 or GLI1 knockdown reduced promoter binding of GLI1 and STAT3, respectively. Finally, chromatin-capture PCR confirmed long-range co-regulation of SOCS2 and FLT3 through changes in promoter conformation. These findings define a mechanism whereby SULF2 drives HCC by stimulating formation of a GLI1-STAT3 transcriptional complex.

Expression of PIM-1 in salivary gland adenoid cystic carcinoma: Association with tumor progression and patients' prognosis.

Pim-1 proto-oncogene, serine/threonine kinase (PIM-1) phosphorylates a series of substrates to exert its oncogenic function in numerous malignancies. The present study investigated the clinical significance of the PIM-1 protein, apoptosis status and apoptosis-associated proteins, including forkhead box O3a (FOXO3a), B cell lymphoma-2 (BCL-2) and BCL-2-associted agonist of cell death (BAD), were investigated in salivary gland adenoid cystic carcinoma (ACC) tissues. PIM-1 expression levels in 4 pairs of ACC tissues and corresponding normal salivary gland tissues were determined by western blot analysis. PIM-1, FOXO3a, BAD and BCL-2 expression levels in 60 ACC tissues were evaluated by immunohistochemistry (IHC). A terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay was performed to detect the apoptosis status of ACC tissues. PIM-1 was revealed to be highly expressed in ACC tissues compared with adjacent normal tissues. IHC staining results demonstrated high expression ratios of PIM-1, FOXO3a, BCL-2 and BAD [33.33% (20/60), 51.67% (31/60), 51.67% (31/60) and 55% (33/60)], respectively, and significant correlations between the expression of PIM-1 and FOXO3a and BCL-2 (P<0.05). Apoptotic rates were significantly associated with PIM-1, FOXO3a, BCL-2 and BAD expression levels (P<0.05). PIM-1 expression levels were significantly associated with tumor size, lymph node involvement, nerve invasion, distant metastasis and weakly associated with tumor node metastasis stage. Kaplan-Meier survival curves revealed that PIM-1 expression level was significantly associated with disease-free survival of patients with ACC (P=0.009). Cox regression multivariate analysis results revealed that histotype, distant metastasis and apoptotic rate were independent prognosis factors for ACC. Assessment of PIM-1 may be useful in investigating the malignant behaviors of ACC and predicting the outcome of patients with ACC.

Mechanisms of resistance of chemotherapy in early-stage triple negative breast cancer (TNBC)

Triple negative breast cancer (TNBC) a clinically aggressive subtype of breast cancer with poor outcomes. Chromosomal instability is a hallmark of many TNBCs, and likely underlies its ability to adapt and rapidly become resistant to chemotherapy. A study of residual disease after neoadjuvant chemotherapy have identified biological mechanisms driving this resistance to chemotherapy. Copy number amplifications such as MCL1, MYC and JAK2, as well as PTEN deletions or mutations have all been identified at a higher frequency in residual disease, suggesting they may play a role in de novo or acquired chemotherapy resistance. Increased copy number and expression of the PIM1 proto-oncogene in TNBC has also been identified as a new target of chemotherapy resistance. However, given the genomic instability and subclonal nature of driver mutations in TNBC, single agent targeted therapy is unlikely to be effective. Lately immune evasion has also been identified as another key characteristic of poor prognostic and chemo-resistant primary TNBCs. Combinations of checkpoint inhibition with targeted therapy and/or chemotherapy are currently being investigated.

PIM1 kinase regulates cell death, tumor growth and chemotherapy response in triple-negative breast cancer.

Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1. Treatment with the pan-PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.

Exploring the IL-21–STAT3 Axis as Therapeutic Target for Sézary Syndrome

Sézary syndrome is an aggressive cutaneous T-cell lymphoma. The malignant cells (Sézary cells) are present in skin, lymph nodes, and blood, and express constitutively activated signal transducer and activator of transcription (STAT)3. STAT3 can be activated by IL-21 in vitro and the IL-21 gene itself is a STAT3 target gene, thereby creating an autocrine positive feedback loop that might serve as a therapeutic target. Sézary cells underwent apoptosis when incubated with Stattic, a selective STAT3 inhibitor. STAT3 activation in Sézary cells did not affect expression of the supposed anti-apoptotic STAT3 target genes BCL2, BCL-xL, and SURVIVIN, whereas expression of (proto)oncogenes miR-21, TWIST1, MYC, and PIM1 was significantly increased. CD3/CD28-mediated activation of Sézary cells induced IL-21 expression, accompanied by STAT3 activation and increased proliferation. Blocking IL-21 in CD3/CD28-activated cells had no effects, whereas Stattic abrogated IL-21 expression and cell proliferation. Thus, specific inhibition of STAT3 is highly efficient in the induction of apoptosis of Sézary cells, likely mediated via the regulation of (proto)oncogenes. In contrast, blocking IL-21 alone seems insufficient to affect STAT3 activation, cell proliferation, or apoptosis. These data provide further insights into the pathogenic role of STAT3 in Sézary syndrome and strengthen the notion that STAT3 represents a promising therapeutic target in this disease.

Pim1 and Myc reversibly transform murine precursor B lymphocytes but not mature B lymphocytes

The proto-oncogenes Myc and Pim1, which are deregulated in many types of cancers, are known to cooperate in B lymphoma development. Here we show that overexpression of retrovirally transduced, doxycycline-inducible Myc alone in IL-7-deprived, growth-arrested pre-B cells enhanced cell cycle entry without impairing apoptosis. Overexpression of Pim1 decreased apoptosis, but had no effect on cell cycle entry. Co-expression of Pim1 and Myc inhibited apoptosis and led to IL-7-independent proliferation of the transduced pre-B cells in vitro, while blocking their differentiation to IgM(+) immature cells. Transplantation of Pim1/Myc overexpressing pre-BI cells into B-cell-deficient mice expanded the pre-B-cell compartments up to 100-fold within 4-8 weeks. Transformation remained dependent on the expression of both oncogenes, as removal of doxycycline in vitro and in vivo terminated proliferation and induced differentiation to IgM(+) B cells. In contrast, Pim1/Myc-transduced mature B cells that developed from the oncogene-transduced pre-BI cells in the absence of oncogene overexpression in vivo were not capable of long-term proliferation after induction of Pim and Myc overexpression, neither in vivo nor in vitro, neither with nor without stimulation by polyclonal activators.

Relapsed MALT Lymphomas Are Associated with a Restricted VH3-30.3 Immunoglobulin VH Gene Repertoire and Are Targeted by Aberrant Somatic Hypermutation

Mucosa associated lymphoid tissue (MALT) type lymphomas are B-cell neoplasms that develop out of a reactive infiltrate; Sjoegren syndrome, Hashimoto thyroiditis, and Helicobacter pylori gastritis provide the pathogenetic background. Direct antigen stimulation through surface immunoglobulin (Ig) molecules may be playing an important role in the development of MALT lymphomas. In rare cases MALT lymphomas may undergo transformation into extranodal diffuse large B-cell lymphomas (eDLBCL). Previous reports suggested salivary gland MALT lymphomas expressed a restricted Ig VH gene repertoire with over use of VH1-69 gene segments. Because knowledge about the VH gene used by MALT lymphoma of gastric and extragastric origin and by eDLBCL is limited, we sequenced the VH genes from 11 MALT lymphomas [5 of gastric and 6 of extragastric origin (3 salivary gland, 3 thyroid)] and from 10 eDLBCL, all arisen from MALT lymphoma and still exhibiting a low grade component [= so called “transformed MALT lymphoma”: 5 of gastric and 5 of extragastric origin (3 salivary gland and 2 thyroid)]. MALT lymphomas used gene segments of the VH1 (1–69: 2 salivary gland and 1 thyroid), VH3 (3–30.3: 1 thyroid), VH4 (4–34: 1 thyroid, 1 salivary gland and 2 gastric), VH5 (5–51: 2 gastric) and VH6 (6-1: 1 gastric) families. Extranodal DLBCLs used segments derived from VH1 (1–69: 1 gastric and 1 salivary gland), VH2 (2–70: 1 thyroid), VH3 (3–23: 1 gastric; 3–30: 1 gastric; 3–30.3: 1 gastric and 2 salivary glands) and VH4 (4–34: 1 gastric) families as shown in table 1. The VH1-69 gene segment was found in 3 of 6 extragastric MALT lymphomas (2 salivary glands and 1 thyroid), in one gastric and one salivary gland eDLBCL but in no gastric MALT lymphoma. Further, 4 of the 21 lymphomas relapsed, 3 eDLBCL and one MALT lymphoma – and remarkably, all of them used the VH3-30.3 gene segment. Comparing the frequency of somatic hypermutation (SHM) of the immunoglobuline locus and aberrant somatic hypermutation (ASHM) of the four proto-oncogenes PAX-5, PIM-1, Rho/TTF and c-MYC between the relapsed (n=4) and non relapsed lymphomas (n=17) a ~2.9 fold (5.8 vs 2.0 × 10−2/bp, p=0.017) and 2.1 fold (0.067 vs 0.032 × 10−2/bp, p=0.027) higher mutation rate for SHM and ASHM in relapsed lymphomas was observed. Also, the AID mRNA relative expression number was 1.7 fold higher in the group of relapsed lymphomas (4.66 vs 2.71, p=0.049). Performing immunohistochemical analysis for AID a significant positive correlation (p=0.01; correlation coefficient (Spearman rho) = 0.794) was observed when comparing protein expression with mRNA levels. These results are consistent with the model in which only certain B cells displaying specific patterns of VH immunoglobuline molecules binding to an as yet undefined antigen together with a highly aberrant somatic hypermutation process are preferentially selected for malignant transformation and determine the natural course of disease.Table1: VH gene sement used by MALT lymphomas and eDLBCLCaseTypeOriginVH geneRelapseGHM2eDLBCLstomach1–69noGHM3eDLBCLstomach3–23noGHM5eDLBCLstomach3-30-3yesGHM7eDLBCLstomach4–34noGHM8eDLBCLstomach3–30noEHM12eDLBCLthyroid6–1noEHM15eDLBCLsalivary gland3-30-3yesEHM16eDLBCLsalivary gland3-30-3yesEHM18eDLBCLthyroid2–70noEHM19eDLBCLsalivary gland1–69noENM14MALTthyroid3-30-3yesENM21MALTsalivary gland1–69noENM29MALTsalivary gland1–69noENM30MALTsalivary gland4–34noENM33MALTthyroid1–69noENM35MALTthyroid4–34noGNM36MALTstomach5–51noGNM37MALTstomach4–34noGNM38MALTstomach6–1noGNM39MALTstomach5–51noGNM41MALTstomach4–34no

New prognostic relevant factors in primary cutaneous diffuse large B-cell lymphomas

There is a growing body of literature that has enhanced our understanding of the biology of primary cutaneous diffuse large B-cell lymphoma (PCDLBCL) including in the context of gene profiling studies. Recent studies have demonstrated an activated proliferation profile associated with leg type lymphoma including overexpression of proto-oncogenes PIM1, PIM2, and cMYC, and the transcription factors MUM1 and OCT2. Although gene profiling is very useful in understanding the molecular basis of diffuse large B-cell lymphoma (LBCL), it is not practical from a routine diagnostic perspective. In this regard, the purpose of the study was to further define an armamentarium of easily applied immunohistochemical stains to accurately prognosticate PCDLBCL. In all, 35 patients with PCDLBCL, 14 of follicle center and 21 of leg type, were analyzed using antibodies against CD5, CD138, BCL2, BCL6, OCT2, MUM1, FOXP1, and cMYC. Findings were correlated with clinical data. All cases stained negative for CD5 and CD138. Both subtypes differed in distinct staining patterns for BCL6, BCL2, OCT2, MUM1, and FOXP1. Staining for BCL2, OCT2, and/or MUM1 was associated with poor, and BCL6 with a favorable prognosis. Expression of cMYC was irrespective of prognosis or subtype, whereas ulceration or primary manifestation on the leg or multiple lesions was indicative for worse prognosis. Case number was a limitation. Discriminating PCDLBCL supports the validity of the World Health Organization/European Organization for Research and Treatment of Cancer classification. To identify risk factors in patients with PCDLBCL we recommend thorough evaluation of clinical presentation and exploratory staining pattern for BCL2, BCL6, MUM1 and OCT2.

Up-regulation of a serine–threonine kinase proto-oncogene Pim-1 in oral squamous cell carcinoma

The Pim-1 proto-oncogene, encoding a serine–threonine kinase, has been found to play an important role in regulating apoptosis, differentiation, proliferation and tumourigenesis. The present study was conducted to assess the importance of Pim-1 in oral tumourigenesis in vivo. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used to study the expression of Pim-1 in oral squamous cell carcinoma (OSCC) and non-cancerous match tissue (NCMT) sampled from the periphery of the tumours. Pim-1 mRNA expression in OSCC was significantly higher than that in NCMT in 36 tissue pairs (1.33 ± 0.41 versus 0.97 ± 0.29, P = 0.03). The percentage of OSCCs exhibiting strong cytoplasmic Pim-1 immunoreactivity was significantly higher than that of NCMT (60% versus 19%, P = 0.007). Pim-1 immunoreactivity is higher in the more differentiated components of a tumour. In around 10% of OSCC cases, Pim-1 immunoreactivity was found in the nucleus as well. These results show novel findings of the up-regulation of Pim-1 expression from NCMT to OSCC. The pathogenetic role of Pim-1 expression in oral tumourigenesis deserves further investigation.

Distinct types of primary cutaneous large B-cell lymphoma identified by gene expression profiling

AbstractIn the European Organization for Research and Treatment of Cancer (EORTC) classification 2 types of primary cutaneous large B-cell lymphoma (PCLBCL) are distinguished: primary cutaneous follicle center cell lymphomas (PCFCCL) and PCLBCL of the leg (PCLBCL-leg). Distinction between both groups is considered important because of differences in prognosis (5-year survival > 95% and 52%, respectively) and the first choice of treatment (radiotherapy or systemic chemotherapy, respectively), but is not generally accepted. To establish a molecular basis for this subdivision in the EORTC classification, we investigated the gene expression profiles of 21 PCLBCLs by oligonucleotide microarray analysis. Hierarchical clustering based on a B-cell signature (7450 genes) classified PCLBCL into 2 distinct subgroups consisting of, respectively, 8 PCFCCLs and 13 PCLBCLsleg. PCLBCLs-leg showed increased expression of genes associated with cell proliferation; the proto-oncogenes Pim-1, Pim-2, and c-Myc; and the transcription factors Mum1/IRF4 and Oct-2. In the group of PCFCCL high expression of SPINK2 was observed. Further analysis suggested that PCFCCLs and PCLBCLs-leg have expression profiles similar to that of germinal center B-cell–like and activated B-cell–like diffuse large B-cell lymphoma, respectively. The results of this study suggest that different pathogenetic mechanisms are involved in the development of PCFCCLs and PCLBCLs-leg and provide molecular support for the subdivision used in the EORTC classification.

Primary diffuse large B-cell lymphomas of the central nervous system are targeted by aberrant somatic hypermutation

AbstractWe have addressed whether aberrant ongoing hypermutation can be detected in the proto-oncogenes PIM1, c-MYC, RhoH/TTF, PAX5, and the tumor-suppressor gene CD95 in primary central nervous system lymphomas (PCNSLs) derived from immunocompetent HIV-negative patients. Nine of 10 PCNSLs analyzed harbored somatic mutations in the PIM1, c-MYC, RhoH/TTF, and PAX5 genes, but not in the CD95 gene, with 8 tumors carrying alterations in at least 2 of these genes. Furthermore, ongoing aberrant mutation was evidenced in a subset of PCNSLs (2 of 3). Although most of the mutations corresponded to base pair substitutions, deletions were also present. The mean mutation frequency was approximately 60-fold lower for these genes compared with the values obtained for immunoglobulin genes in PCNSL. They were increased 2- to 5-fold compared with extracerebral diffuse large B-cell lymphoma (DLBCL). In summary, our data demonstrate aberrant somatic hypermutations at high frequency in the PIM1, PAX5, RhoH/TTF, and c-MYC genes in most PCNSLs. These findings may indicate a pathogenic role for aberrant somatic hypermutation in PCNSL development. In contrast, although mutations were detected in exon 9 of the CD95 gene, the lack of mutations in the 5′ region provides no evidence for the CD95 gene as a target for aberrant somatic mutation.