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Certain subtypes of acute myeloid leukemia (AML) in children have still inferior outcome. One of these AML subtypes has a translocation t(7;12)(q36;p13), always leading to high expression of MNX1 and often to MNX1::ETV6 fusion expression. Here we identified the transforming event in this AML and possible ways to target them. Only MNX1 was able to induce AML in mice, and this was observed using hematopoietic stem and progenitor cells derived from fetal origin but not from adult bone marrow. The restriction in the transforming capacity to cells from fetal liver origin is in concordance with the fact that t(7;12)(q36;p13) AML is mostly restricted to infants. Ectopic expression of MNX1 led to increase of H3K4me1, H3K4me2 and H3K4me3, reduction in H3K27me3, accompanied with changes in genome-wide chromatin accessibility and genome expression, likely mediated through MNX1 interaction with the methionine cycle and different methyltransferases. MNX1 expression resulted in increased DNA damage, depletion of the Lin-/Sca1+/c-Kit+ population and skewing toward the myeloid lineage. These effects, together with leukemia development, was prevented by the S-adenosylmethionine analog Sinefungin. In conclusion, we have shown the importance of MNX1 in leukemia development in AML with t(7;12), supporting a rationale for targeting MNX1 and downstream pathways.

Certain subtypes of acute myeloid leukemia (AML) in children have inferior outcome. One of these has a translocation t(7;12)(q36;p13) leading to aMNX1::ETV6fusion along with high expression of MNX1. Here we identified the transforming event in this AML and possible ways of treatment. Only MNX1 was able to induce AML in mice, with similar gene expression and pathway enrichment to t(7;12) AML patient data. Importantly, this leukemia was only induced in immune incompetent mice using fetal but not adult hematopoietic stem and progenitor cells. The restriction in transforming capacity to cells from fetal liver is in alignment with t(7;12)(q36;p13) AML being mostly seen in infants. Expression of MNX1 led to increased histone 3 lysine 4 mono-, di- and trimethylation, reduction in H3K27me3, accompanied with changes in genome-wide chromatin accessibility and genome expression, likely mediated through MNX1 interaction with the methionine cycle and methyltransferases. MNX1 expression increased DNA damage, depletion of the Lin-/Sca1+/c-Kit+ population and skewing toward the myeloid lineage. These effects, together with leukemia development, was prevented by pretreatment with the S-adenosylmethionine analog Sinefungin. In conclusion, we have shown the importance of MNX1 in development of AML with t(7;12), supporting a rationale for targeting MNX1 and downstream pathways.

Acute myeloid leukemia (AML) results from aberrant hematopoietic processes and these changes are frequently initiated by chromosomal translocations. One particular subtype, AML with translocation t(7;12)(q36;p13), is found in children diagnosed before 2 years of age. The mechanisms for leukemogenesis induced by t(7;12) is not understood, in part because of the lack of efficient methods to reconstruct the leukemia-associated genetic aberration with correct genomic architecture and regulatory elements. We therefore created induced pluripotent stem cell (iPSC) lines that carry the translocation t(7;12) using CRISPR/Cas9. These t(7;12) iPSC showed propensity to differentiate into all three germ layers, confirming retained stem cell properties. The potential for differentiation into hematopoietic stem and progenitor cells (HSPC) was shown by expression of CD34, CD43 and CD45. Compared with the parental iPSC line, a significant decrease in cells expressing CD235a and CD41a was seen in the t(7;12) iPSC-derived HSPC (iHSPC), suggesting a block in differentiation. Moreover, colony formation assay showed an accumulation of cells at the erythroid and myeloid progenitor stages. Gene expression analysis revealed significant down-regulation of genes associated with megakaryocyte differentiation and up-regulation of genes associated with myeloid pathways but also genes typically seen in AML cases with t(7;12). Thus, this iPSC t(7;12) leukemia model of the t(7;12) AML subtype constitutes a valuable tool for further studies of the mechanisms for leukemia development and to find new treatment options.

Carbon nanotubes (CNTs) are a class of carbon based nanomaterials which have attracted substantial attention in recent years as they exhibit outstanding physical, mechanical and optical properties. In the last decade many studies have emerged of the underlying mechanisms behind CNT toxicity including malignant transformation, the formation of granulomas, inflammatory responses, oxidative stress, DNA damage and mutation. In the present investigation, we studied the biodegradation of single-walled carbon nanotubes (SWCNTs) by Cytochrome P450 enzymes (CYP3A4) through using Raman spectroscopy. CYP3A4 is known isozyme accountable for metabolizing various endogenous and exogenous xenobiotics. CYP3A4 is expressed dominantly in the liver and other organs including the lungs. Our results suggest that CYP3A4 has a higher affinity for p-SWNTs compared to c-SWNTs. HEK293 cellular viability was not compromised when incubated with SWNT. However, CYP3A4 transfected HEK293 cell line showed no digestion of c-SWNTs after incubation for 96 h. Cellular uptake of c-SWNTs was observed by electron microscopy and localization of c-SWNTs was confirmed in endosomal vesicles and in the cytoplasm. This is the first study CYP3A4 degrading both p-SWNTs and c-SWNTs in an in vitro setup. Interestingly, our results show that CYP3A4 is more proficient in degrading p-SWNTs than c-SWNTs. We also employed computational modeling and docking assessments to develop a further understanding of the molecular interaction mechanism.

Pediatric acute myeloid leukemia (AML) with the translocation t(7;12)(q36;p13) is a form of childhood leukemia associated with inferior outcome. In the current investigation we have studied the gene alterations that are induced by this chromosomal translocation, a gene fusion MNX1-ETV6 but also ectopic expression of the homeobox gene MNX1. In a murine bone marrow transplantation model, our results showed that ectopic expression of MNX1 was able to induce AML, but not the MNX1-ETV6 gene fusion. However, this transformation to AML was only seen using hematopoietic progenitor cells from fetal liver and not adult bone marrow cells. This was accompanied with an alteration of cell differentiation towards myeloid lineage, increased proliferation and colony formation capacity induced by MNX1. Our data also showed that ectopic expression of MNX1, and to a lower extend MNX1-ETV6 fusion, induced DNA damage and cell cycle arrest, where we saw an increased expression and binding of the MNX1 and the MNX1-ETV6 fusion to the gene CEP164 that has a critical role in cell cycle regulation and DNA damage response, and the transcriptional pioneer factor gene PBX1 that is important in development and differentiation. Furthermore, global histone modifications, specifically H3K4me3 and H3K27me, were induced by MNX1 but also the MNX1-ETV6 fusion. We could also see an enrichment of methylation/methionine proteins from mass spectrometry analysis of proteins associated with MNX1. In conclusion, these results suggest a possible mechanism by which MNX1 mediates its transforming effect through abnormal methylation of proteins that result in changed histone modifications and subsequent dysregulation of gene expression. Our finding that only fetal liver cells were able to transform into AML is also in concordance with the clinical finding that this AML subtype is only found in very young children.

The insulin-like growth factor 1 receptor (IGF-1R) has been suggested to be involved in hepatocyte differentiation. Human hepatocyte cancer cells and stem cells are known to express IGF-1R whereas normal hepatocytes do not. In the present study we optimized a differentiation protocol and verified the different stages by established markers. The expression levels of IGF-1R and major downstream signaling proteins during differentiation from human embryonic stem cells (hESC) to mature hepatocytes were investigated. We could only demonstrate a minor decrease in IGF-1R expression during endodermal differentiation compared to hESC, but declined substantially (>50%) after hepatic lineage commitment during the hepatocyte specification and maturation stages. This downregulation was paralleled by an upregulation of ERK 1/2, AKT and insulin substrate-1. Neither inhibition nor activation of IGF-1R had any essential effect on endoderm differentiation of human embryonic stem cells. Therefore, our data suggest that IGF-1R downregulation may have a regulatory impact after initiation of hepatic lineage commitment.

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-associated tubulin within minutes, indicating that it interfered with microtubule dynamics. These results provide a novel IGF-1R-independent mechanism of antitumor effects of PPP.

Introduction: Non-random cytogenetic aberrations are often involved in the development of AML in children and several aberrations can serve as diagnostic markers, prognosis predictors and impact choice of therapy. In infant AML, a chromosomal translocation t(7;12)(q36;p13) has been found in up to 20-30 % of the cases, making it the second most common genetic aberration in this age group after KMT2A (MLL) rearrangements. Previous studies indicate that this patient group has a dismal prognosis with virtually no event-free survival. Limiting the chances to improve this is the lack of understanding how the t(7;12)(q36;p13) is involved in leukemia development. The translocation leads to a gene fusion MNX1-ETV6 but also to increased MNX1 gene expression. Although both ETV6 and MNX1 are expressed in normal hematopoietic tissues, the role of the fusion protein MNX1-ETV6in the development of AML is not established. Also unclear is whether the driver of leukemogenesis is the fusion itself or the simultaneous overexpression of MNX1. The aim of this study was to assess the transformation capacity and the molecular mechanism of the MNX1-ETV6 fusion and the overexpressed MNX1in vitro and in vivo using murine transplantation models. Material and methods: In a liquid culture system, we introduced the MNX1-ETV6 fusion, MNX1 overexpression, or empty vector into primary murine (C57BL/6) hematopoietic progenitor cells with retroviral transfection. Cells were isolated from either adult bone marrow after 5-FU stimulation, or from fetal liver at E14.5. After enrichment by fluorescence activated cell sorting based on vector co-expressed green/yellow fluorescence protein, transfected cells were used for in vitro experiments and for transplantation into lethally irradiated immunocompetent C57BL/6 mice or sub-lethally irradiated immunocompromised NSGW41 mice. In vitro, cells were assessed with RNA sequencing for gene expression, gamma H2AX assay for DNA double strand brakes, flow cytometry for lineage marker expression, apoptosis and proliferation, and with colony forming unit assay. Results: Upon transplantation, only fetal liver cells transduced with MNX1 or with MNX1-ETV6 fusion were able to induce leukemia in immunocompromised (NSGW41) mice. When MNX1 or MNX1-ETV6 transduced cells were transplanted into immunocompetent mice (C57BL/6) mice, no leukemia development was seen, when either fetal liver or adult bone marrow cells were used for transduction. However, when immunocompromised mice were transplanted with MNX1 or MNX1-ETV6 fusion expressing cells they typically developed signs of disease after 1-2 months and exhibited leukocytosis and elevated blast cells in blood and bone marrow, severe anemia, and enlarged spleens with infiltration of leukemic cells. The cells showed expression of predominantly myeloid markers. In vitro, cells with overexpression of MNX1 or MNX1-ETV6 fusion expression also showed altered lineage differentiation in favor of myeloid differentiation. In addition, MNX1 overexpressing cells, but not MNX1-ETV6 expressing cells, exhibited increased proliferation and colony formation capacity. Both MNX1 overexpressing and MNX1-ETV6 fusion expressing cells showed increased DNA damage as evident from an increased gamma-phosphorylated H2AX in fetal liver and adult bone marrow transduced cells respectively, accompanied with G1 arrest, compared to cells transduced with empty vectors. Both MNX1 and MNX1-ETV6 also led to increased apoptosis in adult bone marrow (3-fold) and to a lesser extent in fetal liver cells (1.5-fold). Results from transcriptome sequencing showed enrichment for specific pathways in G2/M transition of cell cycle in cells transduced by either MNX1or the MNX1-ETV6 fusion. Further investigations to elucidate the molecular mechanisms and pathways through which MNX1 and/or MNX1-ETV6 induce leukemia is ongoing. Conclusions: MNX1 overexpression and MNX1-ETV6 fusion, both characteristics of infant AML with t(7;12)(q36;p13), induced leukemogenic effects in both fetal liver cells and adult bone marrow cells, but could cause a myeloid leukemia only under immunocompromised conditions. This may be of importance for the exclusive prevalence of this AML subtype in young children, with the highest peak during the first six months of life when the immune system is less developed. Disclosures No relevant conflicts of interest to declare.

Introduction Acute myeloid leukemia (AML) is the result of aberrant hematopoietic processes, such as enhanced proliferation, blocked differentiation, and dysregulated apoptosis of hematopoietic stem and progenitor cells, and frequently these changes are initiated by chromosomal translocations in leukemia. Efficient methods for modelling leukemia and to recreate leukemia-associated genetic aberrations, such as chromosome translocations, are therefore crucial for investigating how leukemia is initiated. Today, most such models are murine and usually based on introduction of fusion gene transcripts of interest under the control of a constitutive active promoter using lenti- or retroviral transduction rather than the chromosomal translocation itself. The aim of the current project was to create a human cellular model of a chromosomal translocation that is typically found in AML in children under 24 months of age, the translocation t(7;12)(q36;p13). This translocation has been associated with poor prognosis, and leads to a gene fusion MNX1-ETV6 but also aberrant MNX1 gene expression. Its mechanism for leukemia initiation is so far unknown, mainly due to lack of a suitable experimental model. Material and methods CRISPR/Cas9 was used to reconstruct the genetics of the t(7;12)(q36;p13) rearrangement in human induced pluripotent stem cells (iPSC) while maintaining the genomic architecture and regulatory elements. Ribonucleoprotein (RNP) complex was delivered by lipofection (Nucleofection, Amaxa 4D system) into undifferentiated iPSC (ChiPSC 22, Cellartis). An ATTO550 tag on tracrRNA/RNP complex was used to sort out positive cells by flow cytometry and then seeded as single-cells in 96-well plates. Genomic DNA from the single-cell derived iPSC clones were screened by PCR for the presence of the translocation and positive clones were verified with a FISH probe specific for t(7;12)(q36;p13) (Double Fusion Break Apart probe, Metasystem). RT-qPCR was used to detect and quantify the expression of MNX1-ETV6 fusion and MNX1 transcripts. Differentiation potential was tested with the Trilineage Differentiation and Hematopoietic Kits (STEMdiff, STEMCELL Technologies). Results Using CRISPR/Cas9, we could successfully generate iPSC with the t(7;12)(q36;p13) translocation. The translocation was confirmed using conventional karyotyping and FISH and the mRNA expression of the fusion was confirmed with RT-qPCR. No additional chromosomal aberrations were seen. The t(7;12)(q36;p13) iPSC showed similar growth and differentiation properties as the parental iPSC. They showed propensity to differentiate into all three germ layers, confirming their pluripotent stem cell properties. The potential for differentiation into hematopoietic progenitor cells was shown by expression of CD34+, CD43+ and CD45+. In AML with t(7;12)(q36;p13), MNX1 mRNA expression is increased and this may play a role for leukemia development. In the t(7;12)(q36;p13) iPSC, RT-qPCR indeed showed increased expression of MNX1 expression compared with iPSC without the translocation. This increase of MNX1 was not seen in murine adult bone marrow or fetal liver cells transduced with retrovirus expressing the MNX1-ETV6 fusion. Further characterization of the t(7;12)(q36;p13) iPSC, e.g. whole exome and transcriptome sequencing and engraftment potential in immunocompromised mice (NSG-SGM), is ongoing. Conclusion In summary, we have using CRISPR/Cas9 successfully created a t(7;12)(q36;p13) iPSC line with potential to differentiate into hematopoietic progenitor cells and with gene expression pattern similar to what is seen in human AML samples with the t(7;12)(q36;p13). The introduction of the MNX1-ETV6 fusion in its correct genomic context could recapitulate local gene regulation, making it superior to models based on lenti- or retroviral introduction of fusion genes transcripts. In conclusion, this created cell line will be a valuable tool to study the mechanisms behind t(7;12)(q36;p13) AML, a severe form of AML associated with poor prognosis. Disclosures No relevant conflicts of interest to declare.

Background: The type 1 insulin-like growth factor receptor (IGF-1R) and its signaling components promote cell proliferation, survival, and development of the malignant phenotype. Recently, the IGF-1R has been shown to translocate to the nucleus; however its nuclear functions are not fully elucidated. The proliferating cell nuclear antigen (PCNA) is a nuclear protein that coordinates DNA replication, and is involved in DNA damage repair and in the DNA damage tolerance (DDT) pathways. DDT allows the cell to replicate over polymerase-blocking lesions. DDT mechanisms include translesion DNA synthesis (TLS), and template switching, and the ubiquitination status of PCNA is crucial in these processes. Mono-ubiquitination of PCNA by Rad6 (E2)-Rad18 (E3) activates TLS. The mono-ubiquitin can be further extended though K63-linkages and the non degradative K63 polyubiquitination of PCNA by Ubc13 (E2)-SHPRH/HLTF (E3) leads to template switching. Purpose: The purpose of the present work was to study the functions of nIGF-1R in human embryonic stem cells (hESCs) and in the IGF-1R knockout (igf1r -/-) (R-) and the R- cells overexpressing human IGF1R (R+) mouse embryonic fibroblasts (MEFs), using a mass-spectrometry based approach. Methods and Results: Using subcellular protein fractionation (Qiagen Qproteome Cell Compartment Kit, Qiagen, Germany) and western blot (WB), as well as immunofluorescence, we detected the expression of the nIGF-1R in hESCs, human fibroblasts, MEFs, and in cancer cell lines. To investigate potential functions of nIGF-1R in hESCs, nIGF-1R binding partners were identified using co-immunoprecipitation (Co-IP) and mass spectrometry (MS) (hybrid LTQ-Orbitrap Velos mass spectrometer, Thermo Scientific). Multiple protein candidates were identified including PCNA. IGF-1R-PCNA association was confirmed by Co-IP/immunoblot and by in situ proximity ligation assay (PLA) (OLINK Bioscience, Uppsala, Sweden) to morphologically demonstrate the nuclear localization of the complex. The association of nIGF-1R with PCNA could only be detected in hESCs, MEFs and in human fibroblast cell lines, but not in any of the seven cancer cell lines used. Using a phospho-tyrosine antibody we detected the phosphorylation of PCNA immunoprecipitated from hESCs and R+ but not from R- MEFs or cancer cell lines. Using an in vitro kinase assay, recombinant IGF-1R kinase domain and His-tag recombinant PCNA, we showed that IGF-1R directly phosphorylates PCNA. Using LC-MS/MS and site directed mutagenesis, 3 tyrosine phosphorylation sites (Y60,133,250) were identified. Although PCNA ubiquitination was detected by WB of PCNA immunoprecipitated from R+ MEFs, PCNA protein stability was not affected. The ubiquitination pattern resembled the K63-linked polyubiquitination. Using Co-IP/WB, Ubc13 and SHPRH/HLTF were found to be associated with PCNA in R+ but not in R- cells. Similar results were also achieved for the mono-ubiquitin Rad18 E3 ligase and the main TLS polymerase DNA polymerase. Results were confirmed in hESCs but not in cancer cell lines. Further, PCNA ubiquitination was increased after DNA damage using UV irradiation in R+ MEFs and DNA replication was delayed after methyl methanesulfonate (MMS) treatment, while replication fork stalling in R- cells was highly significant (DNA-fiber labeling assay). Inhibition or knockout of IGF-1R abrogated PCNA phosphorylation, ubiquitination, progress through S-phase of cell cycle, or restart of DNA synthesis after inducing DNA damage. Conclusions: Our results demonstrate a novel role of nIGF-1R in regulating the DNA damage tolerance pathway through PCNA phosphorylation and ubiquitination, potentially maintaining genomic stability in in hESC and in normal fibroblasts. In contrast, nIGF-1R does not interact with PCNA in cancer cell lines. Citation Format: Ahmed Waraky, Yingbo Lin, Eiman Aleem, Olle Larsson. The nuclear IGF-1R regulates DNA damage tolerance through tyrosine phosphorylation of PCNA in human embryonic stem cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B16.