Caride, Ariel J.

Abstract Background Malignant peripheral nerve sheath tumors (MPNSTs) can arise from atypical neurofibromas (ANF). Loss of the polycomb repressor complex 2 (PRC2) is a common event. Previous studies on PRC2-regulated genes in MPNST used genetic add-back experiments in highly aneuploid MPNST cell lines which may miss PRC2-regulated genes in NF1-mutant ANF-like precursor cells. A set of PRC2-regulated genes in human Schwann cells (SCs) has not been defined. We hypothesized that PRC2 loss has direct and indirect effects on gene expression resulting in MPNST, so we sought to identify PRC2-regulated genes in immortalized human Schwann cells (iHSCs). Methods We engineered NF1-deficient iHSCs with loss of function SUZ12 or EED mutations. RNA sequencing revealed 1327 differentially expressed genes to define PRC2-regulated genes. To investigate MPNST pathogenesis, we compared genes in iHSCs to consistent gene expression differences between ANF and MPNSTs. Chromatin immunoprecipitation sequencing was used to further define targets. Methylome and proteomic analyses were performed to further identify enriched pathways. Results We identified potential PRC2-regulated drivers of MPNST progression. Pathway analysis indicates many upregulated cancer-related pathways. We found transcriptional evidence for activated Notch and Sonic Hedgehog (SHH) signaling in PRC2-deficient iHSCs. Functional studies confirm that Notch signaling is active in MPNST cell lines, patient-derived xenografts, and transient cell models of PRC2 deficiency. A combination of MEK and γ-secretase inhibition shows synergy in MPNST cell lines. Conclusions We identified PRC2-regulated genes and potential drivers of MPNSTs. Our findings support the Notch pathway as a druggable target in MPNSTs. Our identification of PRC2-regulated genes and pathways could result in more novel therapeutic approaches.

Abstract Neurofibromatosis type 1 (NF1) syndrome is an autosomal dominant cancer predisposition syndrome defined by germline deletion of one NF1 allele. Somatic loss of heterozygosity in the remaining NF1 allele in Schwann lineage cells gives rise to benign tumors known as plexiform neurofibromas. These tumors have a high risk of transforming into malignant peripheral nerve sheath tumors (MPNST) through the sequential deletion of CDKN2A/B followed by recurrent mutations in SUZ12 or EED, constituting the inactivation of polycomb repressor complex 2 (PRC2). PRC2 is a transcriptional repressor complex involved in the silencing of several development and differentiation genes. Loss of this complex leads to a depletion in trimethylation marks on Histone H3 Lysine 27 (H3K27me3) and the subsequent gain of acetylation marks on the same lysine residue (H3K27Ac). These molecular hallmarks are shared with diffuse intrinsic pontine gliomas (DIPG) harboring the H3K27M mutation which prevents effective deposition of H3K27me3, thus having similar molecular phenotypes to MPNST. However, the full range of epigenetic consequences of these events are currently not well characterized. Additionally, given that MPNSTs originate from cells of neural crest origin and DIPGs from cells of neural origin, it is not know to what degree epigenetic changes stemming from H3K27me3-loss are general across tumors or are context-dependent based on cell-of-origin. In order to contribute to this effort, the Largaespada lab has engineered Schwann cell lines to harbor deletions in NF1 and either SUZ12 or EED and assayed the H3K27ac landscape of these cells in addition to patient-derived MPNST cell lines. Utilizing the ranked ordering of super enhancers (ROSE) algorithm, we have identified common super enhancers within each MPNST cell line. These super enhancer elements are located nearby a variety of putative oncogenes responsible for the maintenance of cell identity. These elements were then compared to publicly available data from DIPG primary and patient-derived samples to understand common regulatory events driven by loss of H3K27me3 as well as molecular signatures present only in MPNST or in DIPG that may be a result of PRC2 or H3K27M alone. We further characterized these super enhancers by assessing the expression levels of their associated genes to understand their role in transcriptional activation. These efforts provide a roadmap of the super enhancer landscape of both MPNSTs and DIPGs to investigate novel mechanisms of H3K27me3-dependent tumorigenesis while also contributing to knowledge of potential effects of H3K27me3-loss that dependent on the cell-of-origin for each respective tumor. Citation Format: Christopher M. Stehn, Minu Bhunia, Kyle Williams, Alex T. Larsson, Ariel Caride, Jeong-Heon Lee, Tamas Ordog, David Largaespada. Characterizing the super enhancer landscape of H3K27me3 loss in MPNST and DIPG [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B009.

Abstract Background The lymphoma proteome is the phenotypic representation of the underlying genetic and epigenetic makeup of each individual patient's tumor. The proteome is rich in druggable targets and offers a unique opportunity for the hematologist to personalize therapy. We developed a deep proteomic profiling method using ion exchange fractionation and tandem mass spectrometry. In this pilot study, we applied this method to detect differentially expressed proteins in anaplastic large cell lymphoma (ALCL) cell lines with previously known (ALK-positive) and unknown (ALK-negative) therapeutic targets. We then asked whether integrative informatic analysis of these data could be used to predict drug sensitivity in each of the cell lines. Methods To examine reproducibility of our method, proteins were extracted independently from 4 pellets each of FE-PD (ALK-negative) and Karpas 299 (ALK-positive) ALCL cells, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin. Resulting peptides were separated into 6 fractions using strong anion exchange (SAX) chromatography. Peptides in each fraction were analyzed via shotgun proteomics on a QExactive mass spectrometer. Peptide mass spectra (MS/MS) were matched against a RefSeq human protein sequence database using MyriMatch software. Reversed sequences were added to the database to measure identification false discovery rates (FDRs). IDPicker filtered the peptide identifications at 2% FDR. Proteins with at least two unique peptide identifications and five MS/MS matches were considered to be present in the sample. Filtered protein identifications and corresponding spectral counts were used as input to QuasiTel software, which was configured to use proteins with at least one spectrum per biological replicate. Proteins that were significantly differentially expressed (quasi p-value < 0.05) with an absolute log2 fold-change of at least 0.5 fold were loaded into the Ingenuity Pathway Analysis (IPA) software, and a master list of drugs and corresponding gene targets was assembled using PharmGKB database and the Drug-Gene Interaction Database (DGID). The resulting drug-gene target list was merged with the differentially expressed protein identifications. Candidate targets were validated by Western blot and candidate drugs were assessed in viability assays. Results The SAX-LC-MS/MS method identified 10,111 proteins from all replicate analyses of FE-PD and Karpas 299 samples, and 93% of the identified proteome was detected in all 4 replicate analyses. The detected proteome was well represented by key transcription factors, phophatases, kinases, translation regulators and transmembrane regulators. There were 1369 proteins differentially expressed between the 2 cell lines, 709 up regulated in Karpas 299 cell line and 673 up regulated in FE-PD. Differentially expressed proteins also showed consistent expression across the biological replicates (Figure 1). Our integrated approach to identify candidate targets and drugs “rediscovered” ALK in Karpas299 and unexpectedly identified relative overexpression of the IL2-IL2RA-STAT5A-STAT5B network in ALK-negative FE-PD cells. Western blot confirmed these findings. As expected, 50-100 nM crizotinib (ALK inhibitor) decreased Karpas 299 viability (p =0.016) but had no effect on FE-PD (Figure 2). In contrast, 50-100 nM of the experimental STAT5 inhibitor 573108 (EMD Millipore) decreased FE-PD viability (p=0.002) but had no effect on Karpas 299 (Figure 2). Conclusion The lymphoma proteome is complex with 10,000 proteins and contains druggable targets that can be reproducibly identified using SAX-LC-MS/MS. These targets vary across samples and integrated informatic analysis can predict target-drug combinations that have efficacy in an experimental model. These data suggest that SAX-LC-MS/MS could be used to personalize treatment regimens in lymphoma patients. Figure 1: Spectral counts of 1369 differentially expressed proteins between Karpas299 and FE-PD cell lines were normalized and plotted in a heat map. Red color indicates down regulation and yellow color indicates up regulation. Disclosures: No relevant conflicts of interest to declare.

The syncytiotrophoblast represents the primary cellular barrier between maternal and fetal circulations in the placenta. Large amounts of Ca2+ are transported across this barrier by mechanisms that are not clearly understood. To further understand this phenomenon, we examined rat and human placenta by immunohistochemical and protein blotting techniques with a monoclonal antibody raised against the human erythrocyte plasma membrane Ca2+ pump. Immunohistochemistry with this antibody showed specific staining in the human placenta of the basal (fetal facing) surface of the syncytiotrophoblast. In the rat placenta, immunohistochemistry also showed specific staining of the innermost (fetal facing) layer of the trophoblast and the basal surface of the endoderm of the intraplacental yolk sac. In Western blots of placental homogenates and membranes, the monoclonal antibody bound to a 140,000-mol wt band, characteristic of Ca2+ pumps in other tissues. Western blots of isolated basal membranes showed more intense staining than isolated microvillous membranes, confirming the results of the immunohistochemistry. In addition, Ca2+ transport in basal membrane vesicles from human placenta was inhibited by polyclonal antibodies prepared against the erythrocyte Ca2+ pump. We conclude that basal (fetal facing) layers of human and rat placentas contain a high-affinity Ca2+ pump situated to transport Ca2+ from the maternal to the fetal circulation. calcium-magnesium-adenosinetriphosphatase; calcium transport; immunohistochemistry; rat and human placenta Submitted on November 14, 1988 Accepted on March 27, 1989

In our previous study of plasma membrane Ca2+ pump (PMCA) isozymes in the rat kidney, we found that the mRNA coding isoform PMCA3 was detected primarily in the outer medullary zone of rat kidney tissue. We now investigated the location of the mRNAs coding for isoforms PMCA3 and PMCA4 of Ca2+ pump in the nephron segments that are present in outer medullary parenchyma using the method of reverse transcription and polymerase chain reaction. In mRNA extracted from whole dissected outer medulla we detected mRNA encoding three splice variants (a, b, and c) of isoform PMCA3; isoform PMCA4 was found in the outer medulla almost exclusively as variant b. Analysis of mRNA from microdissected tubule segments show that proximal straight tubules (PST), medullary thick ascending limbs, outer medullary collecting ducts, and descending thin limb of Henle's loop (DTL) all contained mRNA for isoform 4b. In contrast, the mRNA encoding isoform 3 was detected exclusively in DTL and not in other nephron segments. The unique presence of isoform 3 in DTL is rather surprising, since the specific role of this nephron segment in vectorial Ca2+ transport or in intracellular Ca2+ signaling is not known. The data suggest that isoform 3 in cells of the DTL may have a hitherto unrecognized specific role in Ca2+ signaling or transport of Ca2+, which is distinct from the role of the isoforms of the PMCA in all other nephron segments.

In an effort to extend our studies on Ca2+ pumps to animal models, we developed a new monoclonal antibody (5F10) prepared against the human erythrocyte Ca2+-Mg2+-adenosinetriphosphatase (ATPase) that recognizes a protein of approximately 140 kDa in rat kidney homogenates. Enzyme-linked immunosorbent assays show that monoclonal antibody 5F10 binds purified Ca2+-Mg2+-ATPase and rat kidney membrane extracts in a concentration-dependent manner. In paraffin-embedded tissue sections, antibody 5F10 binds to an epitope in the basolateral membranes of rat kidney distal convoluted tubule principal cells. The antibody does not bind to intercalated cells. The latter cells were characterized by the presence of large amounts of carbonic anhydrase C. Polyclonal antibodies directed against chick intestinal 28-kDa vitamin D-dependent calcium binding protein (28-kDa CaBP) also bind epitopes in distal convoluted tubule cells, connecting tubules, and portions of collecting duct but not intercalated cells. Western blot and 45Ca blot analysis of renal cytosolic proteins showed that the polyclonal 28-kDa CaBP-directed antibody detects a protein which also binds calcium. Western blot analysis with monoclonal antibody 5F10 shows binding to both the authentic purified erythrocyte Ca2+ pump (approximately 138 kDa) and to tryptic fragments of this pump. Antibody JA3, previously used for staining of human kidney tubules, reacts with a different set of tryptic fragments, showing that the two antibodies are directed against different regions or conformational determinants on the pump molecule. We show that Ca2+-Mg2+-ATPase and 28-kDa CaBP are present in the principal cells of the distal convoluted tubule of the rat and are absent in intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51–75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156–1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719–738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, we could identify both hPMCA4a and hPMCA4b. Heart tissue also showed isoform 4b, and probably 4a. In contrast, kidney and lung showed primarily hPMCA4b. In brain, overlapping bands that did not correspond to either variant of hPMCA4 were detected, and in kidney a band migrating in the same position as hPMCA1b was observed. The distribution of the a and b forms of hPMCA4 at the protein level, as analysed by these antibodies, is consistent with the available data about the abundance of mRNAs for the hPMCA isoforms. The presence of hPMCA4b in all the samples supports the proposed role of this isoenzyme as a constitutive form of the pump.

1. A monoclonal antibody (1G4) was raised against the red-cell Ca2+ pump, and it reacted with the pump, as verified by Western blot analysis and by the e.l.i.s.a. method. 2. At 1 mM-ATP and 10 microM-Ca2+, 1G4 inhibited the activity of the purified Ca2+ pump by 40%. 3. Ca2+ pump inhibition by the antibody was non-competitive with regard to Ca2+, calmodulin and the high-affinity portion of the ATP curve. Thus its mechanism was quite different from that of the antibody previously reported [Verbist, Wuytack, Raemaekers, VanLeuven, Cassiman & Casteels (1986) Biochem. J. 240, 633-640], which partially caused inhibition by competition at the ATP site. 4. Antibody 1G4 reduced the steady-state level of phosphorylated intermediate and increased by 50% the calmodulin-activated p-nitrophenyl phosphatase activity of the pump. 5. The experimental results are consistent with the hypothesis that 1G4 inhibits the Ca2+ pump by decreasing the rate of the transition from the E2 form to the E1 form, causing a higher concentration of E2. 6. Analysis by Western blot of the pattern of cross-reaction of 1G4 after tryptic digestion of the pump showed that this antibody reacts with bands of Mr 90,000, 85,000, 50,000 and 33,000. After chymotryptic digestion, the antibody reacts almost exclusively with a fragment of Mr 105,000 that is fully active but is not responsive to calmodulin. Altogether, the results indicate that 1G4 binds to an epitope involved in the functional properties of the enzyme but which is not related to the calmodulin-binding domain.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.

Plasma membrane calcium pumps differentially affect the pattern of calcium signals.