Chromosome 22q11 Research Articles

Bioinformatics analysis of miRNAs in the neuroblastoma 11q-deleted region reveals a role of miR-548l in both 11q-deleted and MYCN amplified tumour cells

Neuroblastoma is a childhood tumour that is responsible for approximately 15% of all childhood cancer deaths. Neuroblastoma tumours with amplification of the oncogene MYCN are aggressive, however, another aggressive subgroup without MYCN amplification also exists; rather, they have a deleted region at chromosome arm 11q. Twenty-six miRNAs are located within the breakpoint region of chromosome 11q and have been checked for a possible involvement in development of neuroblastoma due to the genomic alteration. Target genes of these miRNAs are involved in pathways associated with cancer, including proliferation, apoptosis and DNA repair. We could show that miR-548l found within the 11q region is downregulated in neuroblastoma cell lines with 11q deletion or MYCN amplification. In addition, we showed that the restoration of miR-548l level in a neuroblastoma cell line led to a decreased proliferation of these cells as well as a decrease in the percentage of cells in the S phase. We also found that miR-548l overexpression suppressed cell viability and promoted apoptosis, while miR-548l knockdown promoted cell viability and inhibited apoptosis in neuroblastoma cells. Our results indicate that 11q-deleted neuroblastoma and MYCN amplified neuroblastoma coalesce by downregulating miR-548l.

Increased Frequency of Mosaic Chromosomal Alterations in Peripheral Blood Samples of Sub-Saharan African Children with Endemic Burkitt Lymphoma

Introduction: Burkitt lymphoma (BL) is an aggressive B-cell lymphoma with reciprocal IG∷MYC translocations and accounts for 50-75% of childhood cancers in sub-Saharan Africa. Mosaic chromosomal alterations (mCAs) are large, somatically acquired structural chromosomal alterations that are clonal and observed in detectable fractions of circulating mononuclear cells. mCAs have been linked to increased hematologic cancer risk, but their association with BL, a mononuclear B cell tumor, is unknown. We hypothesized that the frequency and distribution of mCAs in peripheral leukocytes would be elevated in children with BL, potentially serving as a biomarker of chromosomal instability relevant to BL risk. Methods: We investigated peripheral leukocyte DNA extracted from children enrolled in the Epidemiology of Burkitt Lymphoma in East African Children and Minors (EMBLEM) study (2010-2016) and the Infections and Childhood Cancer case-control study conducted in Malawi (2005-2010). We used genome-wide single nucleotide polymorphism (SNP) array data to investigate the frequency, type, and distribution of mCAs >2Mb in size detected in peripheral leukocytes of 931 pediatric BL cases with 3,645 healthy children in Uganda, Tanzania, and Kenya, and with 177 non-BL pediatric cancers in Malawi. Eagle2 and SHAPEIT4 software were used for phasing to infer haplotypes for SNP array data and WGS data. MoChA (v2014-05-14) was used to detect mCAs. Parallel analysis using similar methods was performed on whole genome sequences (WGS) from paired tumor-normal blood samples from two Ugandan BL patients from the BL Genome Sequencing Project (BLGSP) and from paired BL- and lymphoblastoid-derived cell lines. Results: Most samples (92%) were from the EMBLEM study, whereas 8% were from Malawi. The mean age of the BL cases and controls was similar (~7.4 years in both, P=0.60). Table 1 shows detected autosomal mCAs for BL cases and controls by country. Overall, a total of 250 autosomal mCAs were detected in 78 (8.4%) of the 931 BL cases. By comparison, a total of 188 autosomal mCAs were detected in 131 (3.4%) of the 3822 controls. Autosomal mCAs were detected in cell fractions ranging from 0.5% to 99.2% (Figure S1). The average number of autosomal mCAs per person (i.e., total of mCAs detected/number of individuals with mCAs detected) was higher in the BL cases compared to controls (3.21 [SD 3.24] versus 1.44 [SD 2.31], P-value=4.50×10-5. The higher frequency of mCAs in blood samples of children with BL compared to controls was independent of age, sex, P falciparum status, and country (OR=2.8, 95% CI=2.06-3.81, P-value=5.70×10-11, Table 1. mCAs on the X chromosome in girls were also associated with BL (5% versus 1.7%, OR=3.12, 95% CI=1.47-6.29, P-value=1.65×10-3). We did not observe an association for mCAs on the Y chromosome in boys. Analysis of paired tumor-normal samples confirmed the presence of the IG∷MYC translocation in tumor, but the same pipelines did not detect the translocation in the corresponding normal blood sample. In informative tumor-normal cases, some mCAs were only detected in tumor, others were detected in both tumor and normal blood, whereas others were detected mCAs in normal only suggesting some mCAs in blood could arise from pre-BL clones. mCAs only observed in BL cases were predominantly gains on chromosome 1q and 8 (Figure 1) which contain major drivers of Burkitt lymphomagenesis including PLEKHO1, MCL1, PSMB4, ILF2, HAX1, ATP8B2 and CKS1B.Conclusion: We report a high frequency of mCAs in healthy sub-Saharan African children and even higher frequency in pediatric BL cases. The mCAs in children with BL clustered on chromosomes 1q and 8 in regions that overlap with several significantly mutated BL genes, suggesting that mCAs may be potential biomarkers important in the natural history of BL. Using paired tumor-normal sample WGS data, we report both similar and unique mCAs in the paired samples suggesting pre-BL clones in blood. These methods are scalable to large cohorts with SNP array data to investigate health effects associated with mCAs in Africa. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Online Platform for SAMD9 and SAMD9L Variant Annotation and Phenotype Correlation

SAMD9 and SAMD9L (SAMD9/9L) syndromes are new bone marrow failure (BMF) disorders with multisystem involvement (neurological, endocrine, urogenital, and others) and a propensity to develop myelodysplasia (MDS) with monosomy 7 (-7). The SAMD9/9L genes are located on chromosome 7q and are implicated in antiviral immunity, cellular growth, and protein translation. Since the discovery of germline SAMD9L mutations (SAMD9Lmut) in patients with ataxia-pancytopenia and SAMD9 mutations (SAMD9mut) in MIRAGE syndrome, several reports expanded the clinical spectrum to non-syndromic familial MDS, CANDLE syndrome, B-cell aplasia, and steroid-resistant nephrotic syndrome. Because SAMD9/9Lmut proteins enhance the growth-inhibitory effect of wildtype proteins, they are considered gain-of-function (GOF). Clonal rescue hematopoiesis is common: loss or inactivation of the mutant allele occurs by developing -7 clones, via compensatory somatic SAMD9/9Lmut, or uniparental isodisomy 7q (UPD7q). Major challenges for variant interpretation are: i) most germline mutations are novel, ii) ~70% of the mutations are classified as variants of uncertain significance (VUS), iii) the expressivity and penetrance are variable, iv) the associated phenotypes are still emerging (Sahoo, Nat Med 2021). To address these obstacles, we established a comprehensive database of SAMD9/9L variants with detailed phenotype ascertainment. We accumulated reports of SAMD9/9Lmut from literature until July 2022 (n=223) and unpublished cases in our registries (n=83). We extracted patient phenotypes, categorized them anatomically and physiologically, and evaluated correlation with genotypes and somatic events. Germline mutations were mapped using ProteinPaint (Fig 1). Genotypes: We assessed 306 cases with 187 unique SAMD9/9Lmut. For SAMD9 genotype 161 patients carried 122 unique germline mutations with R1293 (n=7), R982 (n=6), and E974 (n=6) discerned as frequently mutated amino acid (aa) positions. In comparison, for SAMD9L genotype, 145 SAMD9L patients had 65 unique mutations, and 3 aa residues were recurrently affected in one-third of the patients: R986 (n=22), R1281 (n=12), and H880 (n=12). Phenotypes and clinical status: Hematological abnormalities were described in 92.5% (149/161) of SAMD9mut and 83% (121/145) of SAMD9Lmut cases. The leading non-hematologic phenotypes for SAMD9mut were genitourinary (47.6%, 51/107), endocrine (36.4%, 39/107), gastrointestinal (35.5%, 38/107), neurological (20.5%, 22/107), and cardiac (11.2%, 12/107); in SAMD9Lmut patients, neurological abnormalities were predominant and found in 36% (42/116) of patients, followed by immunological abnormalities (15.5%, 18/116), while typical MIRAGE phenotypes were also observed (n=2). Within published SAMD9mut or SAMD9Lmut cases, 53% (52/98) or 75.0% (87/116) were alive at last follow-up, respectively. Of note, 63% (29/46) of the deceased SAMD9mut patients had full MIRAGE syndrome spectrum. Clonal rescue hematopoiesis: Somatic genetic rescue (SGR) with UPD7q was reported in 11.7% (26/223) of patients (SAMD9mut 5.6%; SAMD9Lmut 17.2%), SGR with somatic SAMD9/9Lmut in 22.0% (49/223) of cases (SAMD9mut 17.8%; SAMD9Lmut 25.9%), while -7 clones were present in 46.6% (104/223) of the cohort (SAMD9mut 50.5%; SAMD9Lmut 43.1%). UPD7q was more frequent in SAMD9Lmut compared to SAMD9mut (p<0.01). Moreover, 60% (24/41) of SAMD9Lmut cases with SGR (UPD7q and somatic SAMD9/9Lmut) were associated with germline mutations affecting only 4 aa residues (H880, R986, R1281, and V1512). Lastly, we defined the population minor allelic frequency (MAF) thresholds for germline mutations based on the presence of -7 clones and adaptive SGR. We found that specific MAF thresholds were lower than the generic 0.1% and gene-specific: for -7 vs SGR in SAMD9 (0.002 vs 0.0004)and SAMD9L(0.01 vs 0.002) (Fig. 1). Our study (www.stjude.org/samd9) represents the first effort to develop a genotype-phenotype database of SAMD9/9L syndromes which will allow investigators to deposit variants and compare them with published and unpublished data. In newly discovered predisposition syndromes, databases like the one presented here, will be instrumental in gathering crucial clinical data for variant interpretation, identify new phenotypic and prognostic correlates, and improving patients’ clinical care. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transient Monosomy 7 Is a Rare Event in Young Children with SAMD9L Syndrome

Monosomy 7 and deletion 7q are among the most common chromosomal aberrations in pediatric myeloid malignancies and generally associated with an increased risk of rapid progression to advanced neoplasia, warranting allogeneic hematopoietic stem cell transplantation (HSCT). However, several cases of transient monosomy 7 and spontaneous hematologic recovery upon loss of monosomy 7 in young children have been described in the literature. In several cases, germline mutations in sterile alpha-motif domain-containing protein 9 (SAMD9) and SAMD9-like (SAMD9L), both located on chromosome 7q, have been identified. Gain of function (GOF) mutations in SAMD9 and SAMD9L cause phenotypically overlapping syndromes that predispose to pediatric myelodysplastic syndrome (MDS) and are associated with non-hematological anomalies and variable immunodeficiency. GOF SAMD9/9L mutations inhibit cell proliferation and reduce cell survival of hematopoietic stem and progenitor cells (HSPCs). This may exert strong selection pressure on HSPCs to acquire somatic aberrations that improve their competitive fitness. To no surprise, a distinctive feature of SAMD9/9L syndromes is their propensity for somatic genetic reversion, including somatic loss of function mutations in SAMD9/9L in cis, uniparental isodisomy 7q, and non-random loss of chromosome 7 or 7q of the mutant allele. Multiple hematopoietic subclones with clone-defining somatic mutations can exist in the bone marrow and their composition may change over time due to the exceptionally high plasticity of hematopoiesis in young children. Consequently, monosomy 7 may spontaneously disappear in young patients with SAMD9/9L syndromes. Earlier observations had suggested that somatic compensatory mutations may be more frequent in SAMD9L than in SAMD9 mutated patients. Here, we investigate the natural history of young children with SAMD9L syndrome and monosomy 7. We report on seven patients with SAMD9L syndrome and monosomy 7 presenting before the age of 5 years in whom an initial surveillance strategy was electively chosen rather than timely allogeneic HSCT. All patients presented with refractory cytopenia of childhood with monosomy 7 at a median age of 1.0 years (range: 0.4-3.1 years). Six patients had an additional non-hematologic phenotype, including prematurity (P2, P5), small for gestational age (P1, P2, P7), cerebellar atrophy and developmental delay (P1), bilateral cleft lip and palate (P2), macrocephaly (P3, P6), shorter thumbs (P6), and big eye bulbs and dystrophy (P7). Immunodeficiency was present in five patients ranging from hypogammaglobulinemia (P1, P2, P5, P6, P7) to B and NK cell deficiency patients (P2, P5, P6, P7). At a median follow-up of 35 months (range: 10-51 months), allogeneic HSCT had been employed in four patients for severe neutropenia and/or bacterial infection (P1, P7), persistence of monosomy 7 beyond the age of 5 years (P5) or disease progression to MDS with excess blasts (P2). One patient (P2) suffered transplant-related mortality, while the remaining patients were alive at last follow-up. Three patients did not require definitive treatment. Two of the three patients had complete loss of the monosomy 7 clone with concomitant recovery of blood counts (P4, P6), a third patient had normalized blood counts, but cytogenetic and molecular studies at follow-up were missing (P3). Secondary somatic mutations were detected in six patients. Five patients carried somatic SAMD9L mutations in cis (P2, P3, P4, P6, P7). Two patients with spontaneous loss of monosomy 7 carried large somatic SAMD9L subclones that were stable over time and presumably maintained hematopoiesis (P4, P6). In addition, two patients carried somatic mutations in oncogenic driver genes (P2, P5). P2, who developed MDS-EB 14 months after diagnosis, had two RUNX1 mutations at the time of progression. P5 had one RUNX1 and one EZH2 mutation, but without evidence of disease progression. Our results suggest that loss of monosomy 7 can occur in young patients with SAMD9L syndrome but is a rare event. If a watch and wait strategy is chosen, close monitoring with frequent bone marrow examinations including SAMD9L sequencing, cytogenetics and search for somatic oncogenic events is advised. Importantly, presence of severe infections, severe neutropenia, increase in blast percentage, and persistence of monosomy 7 beyond the age of 5 years should prompt timely allogeneic HSCT.

Germline SAMD9/9L MDS Predisposition Syndromes Are Characterized By Complex Clonal Architecture and Lineage-Specific Escape Mechanisms Including Somatic Genetic Rescue in T and B Lymphocytes

Inherited bone marrow failure (BMF) and MDS predisposition syndromes have an intrinsic defect resulting in loss and/or dysfunction of hematopoietic stem and progenitor cells (HSPC). HSPC clones can escape this process by acquiring gene- or genome-level genetic changes (compensatory mutations or uniparental disomies (UPD)) thereby increasing cellular fitness. This process, referred to as somatic genetic rescue (SGR) has been shown to occur in >70% of patients with gain-of-function mutations in SAMD9 and SAMD9L (SAMD9/9L) genes, which are a common cause of early onset BMF and pediatric MDS with monosomy 7. It is unknown how SGR arises and in which cells it originates. Furthermore, little is known about the epistatic interactions of various rescue events. We aimed to address these questions by utilizing a newly established single cell (sc)-proteogenomic platform that can simultaneously detect single nucleotide variants (SNVs) and copy number variation (CNVs) in addition to protein expression. We applied this technology to study clonal architecture and cellular origins of SGR lesions in a cohort of clinically annotated SAMD9/9L-mutated (SAMD9/9Lmut) patients. We also asked if monosomy 7, traditionally assessed by karyotyping, can be also detected in peripheral blood (PB) of affected patients. We first performed whole exome and panel sequencing to characterize 67 germline SAMD9/9Lmut patients. Guided by this data, we designed a sc-proteogenomic panel with 69 amplicons specific to variants in SAMD9/9L and leukemia driver genes, and 250 amplicons covering high-frequency SNVs on chromosome 7q. For validation of initial findings, we developed a second panel targeting full coding regions of SAMD9/9L genes, and additional 7p SNVs allowing for a precise delineation of uniparental isodisomy 7q (UPD7q). We included oligonucleotide-conjugated antibodies specific to major hematopoietic antigens, including CD19, CD3, CD11b, CD38 and CD34. Using this panel, we analyzed 22 samples (15 bone marrow (BM) and 7 PB) of 10 patients enrolled in local biobanking protocols. On average, 3,845 high-quality cells were genotyped per sample, and we robustly identified SNVs, monosomy 7 and UPD7q changes in single cells. Somatic rescue SAMD9/9Lmut were found only in clones with normal (diploid) chromosome 7, UPD7q (containing two wild type SAMD9/9L alleles after mitotic recombination event) was a stand-alone clone, while monosomy 7 (SAMD9/9Lmut lost) occurred alone or together with leukemia driver mutations. We also detected lesions that were previously missed using routine clinical sequencing in 3 patients (somatic SAMD9/9Lmut or UPD7q). Most patients had branching clonal architecture (i.e., independent monosomy 7, UPD7q, and somatic SAMD9/9Lmut), while linear trajectories were rare (i.e., monosomy 7 à + leukemia driver mutation). Serial assessment of 4 patients with stable hematology (3 with monosomy 7 and 1 with an additional somatic SAMD9/9Lmut) for a period of up to 4.8 years, revealed stable clonal populations without acquisition of additional driver mutations. Quantification of clones in paired BM and PB in 4 patients demonstrated equal frequencies of monosomy 7 and somatic SAMD9/9Lmut in both compartments, demonstrating the utility of this assay in surveillance. Our findings were confirmed by sorting BM cells into different lineages followed by sequencing, error-corrected sequencing, and ddPCR. Finally, analysis of protein expression data revealed that while monosomy 7 is present exclusively in cells with myeloid phenotype, the SGR events can arise in lymphocyte lineage only; 4 patients carried clones with somatic SAMD9/9Lmut in CD3+ lymphocytes, and 2 patients had UPD7q clones in either CD3+ or CD19+ lymphocytes. These findings provide clues why most of the SGR events in patients do not result in long-lasting clinical remission since the genetic rescue can be restricted only to certain hematopoietic lineages. In conclusion, single cell proteogenomic analysis is useful for the understanding the mechanisms of clonal evolution in SAMD9/9L syndromes. This method also offers the opportunity for less invasive surveillance of karyotype abnormalities from peripheral blood in patients who are at high risk for MDS progression.

The Molecular Pathophysiology of Ineffective Erythropoiesis at Single Cell Resolution

The anemias of myelodysplastic syndrome (MDS) and Diamond Blackfan anemia (DBA) are generally macrocytic, always reflect ineffective erythropoiesis, and yet result from diverse genetic mutations. To delineate the shared mechanisms that lead to the death of maturing erythroid cells, we studied single marrow cells from MDS-5q patients, DBA patients, and normal individuals with CITE-seq (antibody barcoding of cell surface proteins) and RNA sequencing. After testing several approaches, we opted to align transcriptomes with SCVelo pseudotime analysis, which includes incompletely processed mRNAs, since these are highly prevalent and regulated during erythropoiesis. We identified 38,296 erythroid lineage cells then plotted CITE-seq protein quantitation data versus the velocity pseudotime to identify BFU-E through orthochromatic erythroblasts based upon the sequential CD117, CD36, CD71, and GlyA expression levels, using well-established criteria. The analysis identified two distinct transcriptional trajectories. These diverge during the CFU-E stage, following upregulation of the transferrin receptor, CD71, and initiation of iron import, and right after upregulation of ALAS2, the first and rate-limiting step of heme synthesis. The normal (healthy) trajectory (trajectory A) contains cells at all stages, including the late stages of erythroid maturation, and there is high expression of mitochondrial oxidative metabolism and erythroid differentiation pathway genes. In contrast, the second trajectory (trajectory B) ends abruptly near the start of terminal erythroid differentiation as defined by cell surface proteins, and highly expresses gene pathways associated with excess heme and with apoptosis. There is loss of mitochondrial gene expression, premature loss of nuclear gene expression (increased % mitochondrial genes), and elevated levels of BAX and other apoptosis markers, implying that this is a death pathway. The significant increase in expression of genes in the heme-responsive dataset defined by Liao et al (Cell Rep. 2020. 31:107832) in trajectory B cells along with the pathway analysis, suggests that DBA and MDS-5q erythroid cells died from heme toxicity, a finding supportive of our earlier work (Yang et al. Sci Transl Med 2016. 8:338ra67). Cells along each trajectory were present in all samples but at different frequencies: 57±4% of MDS-5q, 73±2% of DBA, and 24±12% of normal cells followed trajectory B. That 24±12% of normal cells followed trajectory B was an unexpected finding, and indicated that heme might regulate the ineffective vs. effective differentiation of normal, as well as DBA and MDS-5q cells. As a corollary, drugs or interventions decreasing heme synthesis might modulate trajectory choice and be therapeutically beneficial. Also notably, fate decisions occur well before cell death, allowing time for therapeutic intervention. In addition, since we can independently track the fate and transcriptomes of cells with an intact chromosome 5q vs. cells with chromosome 5q deletion, these experiments provide insight into why anemia occurs early in MDS-5q (i.e., at presentation) often only 50-75% of the marrow is replaced by neoplastic cells. The analysis revealed both 5q- and 5q+ cells show early upregulation of HIF1A targets, ROS response, cell cycle regulation and spliceosome pathways and down regulation of erythroid differentiation and mTOR signaling pathways, indicating both 5q- and 5q+ cells share common pathologies. Additionally, the 5q- cells upregulate p53 pathways. The fact that the 5q+ cells are not normal implies that the presence of neoplastic 5q- cells influence the erythroid differentiation of the 5q+ cells. That they too have transcriptomic findings attributable to excessive heme may suggest a cell-extrinsic role for heme toxicity, which has led us to study heme processing by the central macrophage of erythroblastic islands (EBIs). Initial studies suggest EBI macrophage dysfunction contributes to the poor differentiation of 5q+ cells. This abstract includes data available on the medRxiv preprint server. https://doi.org/10.1101/2022.08.01.22278133

Cytogenetics in Fanconi Anemia: The Importance of Follow-Up and the Search for New Biomarkers of Genomic Instability.

Fanconi Anemia (FA) is a disease characterized by genomic instability, increased sensitivity to DNA cross-linking agents, and the presence of clonal chromosomal abnormalities. This genomic instability can compromise the bone marrow (BM) and confer a high cancer risk to the patients, particularly in the development of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). The diagnosis of FA patients is complex and cannot be based only on clinical features at presentation. The gold standard diagnostic assay for these patients is cytogenetic analysis, revealing chromosomal breaks induced by DNA cross-linking agents. Clonal chromosome abnormalities, such as the ones involving chromosomes 1q, 3q, and 7, are also common features in FA patients and are associated with progressive BM failure and/or a pre-leukemia condition. In this review, we discuss the cytogenetic methods and their application in diagnosis, stratification of the patients into distinct prognostic groups, and the clinical follow-up of FA patients. These methods have been invaluable for the understanding of FA pathogenesis and identifying novel disease biomarkers. Additional evidence is required to determine the association of these biomarkers with prognosis and cancer risk, and their potential as druggable targets for FA therapy.

Suppression of Multiple Myeloma Via Inhibition of EFNB2 Reverse Signaling

Multiple myeloma (MM) cells display a reciprocal relationship with neighboring niche cells in the bone marrow (BM), particularly vascular endothelial cells (BM ECs). This relationship drives disease initiation, progression, and therapy resistance, but the mechanisms underlying this interaction remain incompletely understood. Here, we show that silencing ephrin B2 (EFNB2) blocked MM cell proliferation and survival in vitro and abrogated MM engraftment and repopulation in vivo. EFNB2 expression is also increased in MM patients with adverse-risk cytogenetics and decreased survival, suggesting an important role for EFNB2 in MM disease progression. To identify novel regulators of MM pathogenesis, we performed transcriptome analyses of ECs from multiple human tissues and analyzed their capacity to promote MM growth. The EPH receptor, EPHB1, was overexpressed in human ECs that support MM expansion in culture. Silencing EPHB1 expression in ECs using siRNA or a lentiviral shRNA abrogated MM cell expansion compared to EC co-cultures treated with non-targeting shRNA and blocked the expansion of MM cells capable of in vivo repopulation in immune deficient mice. Additionally, transgenic Vk*MYC mice, which develop indolent murine MM through activation-induced deaminase-dependent MYC activation in germinal B cells, displayed increased expression of EPHB1 and EPHB4 in BM ECs compared to wild type BM ECs. We found that expression of EFNB2, an EPHB1/4 binding partner, is upregulated in 3 human MM cell lines and in primary MM cell populations from patients, whereas other EPHB1/4 binding partners were either not detected or expressed at a low level. shRNA-mediated inhibition of EFNB2 in MM cells blocked MM cell cycling, leading to a near 70-fold reduction in MM cell growth at day 12 of culture, and blunted MM cell repopulation when transplanted into NSG mice. In order to determine the therapeutic potential of targeting EFNB2 in MM in vivo, we transplanted irradiated NSG mice with MM cells and subsequently administered a single chain variable fragment (scFv) targeting EFNB2 on days +4, +6, +10 and +12. Mice treated with the EFNB2 scFv displayed significantly decreased engraftment of MM cells in vivo compared to control mice. MM reconstitution in vivo. Silencing EFNB2 expression in MM cells downregulated several pathways including those involved in cellular movement, cell death and survival, cellular assembly, cell cycle and DNA repair. Several STAT5 target genes were also downregulated in MM cells treated with shRNA-EFNB2, supporting the notion that STAT5 signaling is activated by ephrin B proteins (Bong et al. Proc Natl Acad Sci. 2007;104(44):17305-17310). Consistent with these findings, EFNB2 overexpression via dCas9-VPR increased MM cell survival, proliferation and STAT5 phosphorylation. Conversely, silencing EFNB2 decreased STAT5 activation, increased MM apoptosis, and decreased MM proliferation. Importantly, STAT5 inhibition in MM cells overexpressing EFNB2 blocked EFNB2-mediated MM cell proliferation, suggesting that EFNB2-mediated MM growth is dependent on STAT5. Overexpressing a mutant form of human EFNB2 with GFP replacing the intracellular cytoplasmic PDZ signaling domain did not result in cell growth comparable to overexpressing the full-length EFNB2. Silencing EPHB4, the main EFNB2 binding partner expressed on MM cells, resulted in significant impairment of growth in wild-type MM cells but not those with full-length EFNB2 overexpression. Updated downstream signaling and in vivo data of these will be presented at the meeting. Interrogation of MM clinical databases, including the Total Therapy studies (Zhan F. et al. Blood. 2006;108(6):2020-2028) and the CoMMpass data (Multiple Myeloma Research Foundation, https://research.themmrf.org/, accessed on 20 October 2021, n = 773 patients), revealed that EFNB2 expression is increased in MM patients with adverse risk cytogenetics, including t(4;14) and gain of chromosome 1q, while lower EFNB2 expression is associated with the more favorable, "standard risk" disease category, hyperdiploid MM. Increased EFNB2 expression is also associated with advanced stage disease and decreased survival in MM patients. Our analyses suggest that EFNB2 signaling represents an important new mechanistic target in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of the Proteasome Subunits PSMB4 and PSMD4 As Novel Targets in Multiple Myeloma Patients Carrying 1q21 Amplification

Gain and/or amplification of the 1q21 (1q21+) region is one of the most frequent secondary cytogenetic abnormalities present in multiple myeloma (MM) patients. Interestingly, it has been reported that the incidence of the amplification of chromosome 1q (Amp1q) in patients with pre-malignant smoldering myeloma (SMM) is about 40%. Several data indicate that the presence of Amp1q is associated with a worse clinical outcome and a poor response to therapies including proteasome inhibitors (PIs). Recently, a study showed that newly diagnosed MM (NDMM) carrying Amp1q receiving carfilzomib-based treatment have an early disease progression and shorter overall survival, compared to those with 1q21 gain. The genes driving amplification of the 1q21 region have not been fully elucidated. This study aims to identify novel targets in the 1q21 region possibly correlated to PIs treatment. We purified CD138+ plasma cells (PCs) by immunomagnetic beads from bone marrow (BM) aspirates. We used a total cohort of 29 MM patients: 11 SMM and 18 NDMM. Fluorescent in situ hybridization analysis was performed on all the patients. 48% (14/29) patients carried 1q21+. A score indicating the number of 1q21 copies was calculated based on the FISH hybridization pattern of each patient. The expression profile of all 29 samples was generated using GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA). The smar package was used to identify differentially expressed genes between 1q21+ and control samples. The expression profile of our cohort showed that the expression of both proteasome subunits, PSMB4 and PSMD4 was significantly upregulated in MM patients Amp1q compared to control patients (PSMB4 p=0.0006; PSMD4p=<0.0001 respectively). Additionally, we found a strong positive correlation between gene expression levels and 1q21 copy number for the proteasome subunits PSMB4 (p=<0.0001, r=0.5631) and PSMD4 (p=<0.0001, r=0.6391). Interestingly, the PSMB4 and PSMD4 expression levels were independent of the disease stage (SMM vs MM) and the expression was driven exclusively by the 1q21 copy number. We evaluated PSMB4 and PSMD4 mRNA and protein expression levels in a 1q21 wild-type MM cell line (OCI-MY5) and a panel of MM cell lines carrying 1q21+. Our results showed that PSMB4 and PSMD4 expression levels were higher in cell lines with Amp1q, following a 1q21 copy number fashion. Subsequently, we assessed the role of PSMB4 and PSMD4 to the sensibility of PI bortezomib in two myeloma cell lines carrying 1q21+. Our results showed that bortezomib treatment downregulates the protein expression of PSMD4 while PSMB4 was unaffected. Moreover, the MTT viability test correlated with the anti-myeloma effect of bortezomib. Furthermore, we generated a JJN3 PSMB4 and PSMD4-knockdown MM cell line using short hairpin RNA (shRNA) lentivectors. An empty vector was used as a control plasmid. Cell viability and apoptosis assays were performed using MTT assay and Apo 2.7 staining. Our functional studies showed that shPSMB4 and shPSMD4 decreased MM cell viability when treated with PI. Moreover, both shPSMB4 and shPSMD4 knockdown cells showed elevated levels of polyubiquitylated proteins, indicating blockade of proteasome-mediated protein degradation. In conclusion, our study identified proteasome subunits PSMB4 and PSMD4 to be significantly upregulated in MM patients carrying 1q21+, correlated with 1q21 copy number but not with disease stage. Therefore, targeting PSMB4 and PSMD4 could be a strategy to treat MM patients with 1q21+.

Combining Plasma Cell Leukemia-like Status with the Second Revision of the International Staging System Improves Risk Classification

Introduction Newly diagnosed multiple myeloma (NDMM) is characterized by a highly variable clinical course, with an overall survival (OS) ranging from less than 3 years to over 10 years. While patients with standard risk disease have benefited significantly from the introduction of novel agents, it remains unclear what the best upfront treatment strategy is for those with high-risk disease. As a result, risk-adapted trial designs (e.g., the MUKnine b OPTIMUM study, NCT03188172) are becoming more common in NDMM and are driving research efforts to improve the identification of high-risk disease. High-risk NDMM can be identified with the recently published Second Revision of the International Staging System (R2-ISS), which combines several conventional risk markers including tumor burden and cytogenetic aberrations that generally reflect increased proliferation and resistance to apoptosis (D'Agostino et al. - J Clin Oncol 2022). We have recently shown that NDMM with high levels of circulating tumor cells can be identified based on a plasma cell leukemia-like (PCL-like) transcriptome that reflects aggressive disease, which is different from the high-risk biology that is recognized by conventional risk models (Hofste op Bruinink et al. - J Clin Oncol 2022). Hence, we hypothesized that combining PCL-like status with the R2-ISS classification would improve prognostic accuracy. Methods Baseline characteristics including serum β2 microglobulin, albumin, lactate dehydrogenase and high-risk cytogenetic markers including deletion of chromosome 17p, t(4;14) and gain/amplification of chromosome 1q were collected from NDMM patients enrolled in the HOVON-65/GMMG-HD4 (EudraCT 2004-000944-26), HOVON-87/NMSG-18 (EudraCT 2007-004007-34), MRC-IX (ISRCTN68454111), Total Therapy 2 (NCT00083551), Total Therapy 3 (A: NCT00081939 and B: NCT00572169) and MMRF CoMMpass studies (NCT01454297) and used to determine the R2-ISS classification. Transcriptomic profiles were generated from CD138-enriched bone marrow tumor cells and used to calculate PCL-like status. For progression-free survival (PFS), an event was defined as either progressive disease or death from any cause. For OS, an event was defined as death from any cause. Hazard ratios (HRs) for PCL-like status, the R2-ISS classification or a combination thereof were estimated using a Cox proportional hazards model stratified by study cohort and corrected for age ≤ 65 years. Two-sided P values < 0.05 were considered statistically significant. Results For a total of 976 NDMM patients of known age, both the R2-ISS classification and PCL-like status could be determined, with a median follow-up time of 53 months. 31% of patients were classified as R2-ISS low (R2-ISS I), 31% as R2-ISS low-intermediate (R2-ISS II), 33% as R2-ISS intermediate-high (R2-ISS III) and 5% as R2-ISS high (R2-ISS IV), whereas 9% were classified as PCL-like MM and 91% as not PCL-like MM (i.e., intramedullary MM, i-MM). PCL-like status was associated with both an inferior PFS (HR, 1.8; 95% confidence interval (CI), 1.4 to 2.3; P = 0.001) and OS (HR, 2.2; 95% CI, 1.7 to 2.9; P < 0.001). This also applied to the R2-ISS classification when comparing R2-ISS IV to R2-ISS I, II and III (HR for PFS, 2.5; 95% CI, 1.9 to 3.4; P < 0.001 and HR for OS, 3.0; 95% CI, 2.2 to 4.2; P < 0.001). PCL-like status retained its prognostic significance in a multivariate model when combined with the R2-ISS classification, both in terms of PFS (HR, 1.8; 95% CI, 1.4 to 2.4; P = 0.001) and OS (HR, 2.2; 95% CI, 1.6 to 2.9; P < 0.001). Of note, 1% of NDMM patients were both classified as PCL-like MM and R2-ISS IV, which translated into a median PFS of 4 months (95% CI, 3 months to not reached) and a median OS of 7 months (95% CI, 4 months to not reached). Patients with PCL-like MM and R2-ISS III (3%) had a comparable median OS to those with i-MM and R2-ISS IV (4%) (35 and 30 months, respectively). Conclusions 1. PCL-like status has independent prognostic value in the context of the R2-ISS classification in NDMM. 2. The presence of both PCL-like MM and R2-ISS IV may confer exceptionally high risk in NDMM, with a median OS of only 7 months. 3. NDMM patients with PCL-like MM and R2-ISS III have a comparable OS to those with i-MM and R2-ISS IV, comprising a median OS of 35 and 30 months, respectively.

Prevalence of t(11;14) Multiple Myeloma in Real-World Setting: Interim Analysis from the Medici Study

Introduction: Multiple myeloma (MM) is the second most common hematologic malignancy characterized by bone marrow infiltration by monoclonal plasma cells (PC). Several well-defined genetic abnormalities have been identified as relevant with the disease. Although novel therapies have improved overall survival most patients with MM will eventually relapse or become refractory (RR). Therefore, there is an emerging need for better therapeutic regimens including personalized medicine based on specific biomarkers. Venetoclax (Ven) is a selective and potent oral B-cell lymphoma-2 (BCL-2) inhibitor that has shown significant antimyeloma activity in patients with t(11;14) RRMM (Kumar et al. Blood 2017; 130 (22): 2401-2409). In the phase 3 BELLINI (NCT02755597) study, patients with t(11;14) RRMM showed improved response rates and progression-free survival when treated with Ven in combination with bortezomib and dexamethasone compared to placebo-bortezomib-dexamethasone (Kumar et al. Lancet Oncol. 2020; 21:1630-1642). These results suggest that t(11;14)-based selection of patients with MM may represent a potential biomarker for venetoclax-based treatment regimens. An ongoing phase 3 study, CANOVA (NCT03539744), is evaluating the combination of Ven and dexamethasone in patients with t(11;14) RRMM compared to standard of care pomalidomide-dexamethasone (Mateos et al. J Clin Oncol 2020; 38: no. 15_suppl) . Understanding the prevalence of t(11;14) MM in a real-world setting will provide important insights for targeted therapy in this patient subpopulation. Therefore, the MEDICI study was designed to evaluate the real-world prevalence of t(11;14) as detected by interphase fluorescence in situ hybridization (FISH) technology in patients with MM. Methods: MEDICI (NCT04721002) is an ongoing, prospective, multicenter minimally interventional study. The primary objective is to determine the prevalence of t(11;14) in both newly diagnosed (ND) and RRMM as determined by interphase FISH analysis of monoclonal bone marrow (BM) PCs. All patients (≥18 years) included in the study signed informed consent. BM aspirates (BMA) were collected as part of standard of care procedures performed at diagnosis and disease relapse. Patients with no BMA sample at the time of diagnosis or confirmation of relapse were excluded from the trial. The primary endpoint was the t(11;14) status of the earliest bone marrow sample collected at initial diagnosis or across subsequent lines of therapies. CD138-enriched BMA samples were evaluated for t(11;14) by FISH (Abbott Molecular). Results: Sixty-two patients were included at the time of the interim analysis (data cut-off 09 March 2022). Median age was 67.5 years, 51.6% were male and 95.2% were Caucasian. Patients with ECOG performance status ≤2/>2/unknown was 83.9%/8.1%/8.1%. ISS stage across patients were stage I (30.6%), stage II (22.6%), stage III (33.9%) and unknown (12.9%) respectively. Del 17p was observed in 6.5% of patients and gain of chromosome 1q in 25.8% of patients. There were 36 and 26 patients with NDMM and RRMM respectively. One patient had longitudinal samples. The overall prevalence of patients with t(11;14) MM was 30.0%, which was similar between NDMM (25.0%) and RRMM (37.5%) patients (Table). The distribution of patients across the observed t(11;14) FISH fusion (F) categories (1F/2F/≥3F) for NDMM was 33.3%/22.2%/22.2% and 44.4%/55.6%/0.0% in RRMM pts (Table). Across lines (L) of therapy, the distribution of t(11;14)+ patients were similar between 1L (25.0%) , 2L (35.7%), and 3L+ (36.4%) (Table). The distribution of t(11;14)+ patients across disease stage was also similar between stage I (31.6%), stage II (28.6%), and stage III (25.0%) respectively. There were 8 patients with unknown stage of disease, 3 among them were t(11;14)+. Conclusion: Interim analysis of MEDICI demonstrated that prevalence of t(11;14) was consistent across ND and RRMM setting and independent of line of therapy and disease stage. This study is currently ongoing, and further updates will be provided upon presentation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Co-Occurrence of 1q21+/1q23+ and t(4;14) Abnormalities with Specific Clone Size Is Predictive of Shorter Response to Transplant in Patients with Multiple Myeloma

Introduction: The presence or absence of recurrent chromosomal abnormalities (CA) as detected by karyotype and/or fluorescence in-situ hybridization (FISH) has emerged as one of the most clinically relevant prognostic factors for patients with newly diagnosed multiple myeloma (MM). The Revised-International Staging System (R-ISS) defined high-risk disease as the presence of at least one of the following: deletion(17p), translocation t(4;14)(p16;q32), or translocation t(14;16)(q32;q23). Amplification or gain of chromosome 1q (1q21+/1q23+) has also been growing in its clinical relevance, with 1q21+ found in approximately 35-40% of patients. As a result, this study evaluated differences in survival outcomes and disease characteristics in patients with both 1q+ (1q21+ or 1q23+) and t(4;14), compared to those patients with only 1q+ or t(4;14). Methods: A retrospective chart review was performed on 243 newly diagnosed MM patients with the cytogenetic abnormalities of interest obtained from established FISH data from bone marrow aspirate samples at diagnosis. Primary endpoints were Progression-Free Survival (PFS) and Overall Survival (OS) after autologous stem cell transplant (ASCT), and Kaplan Meier methods were used to calculate PFS and OS. Secondary endpoints included the identification of disease characteristics between single CA and combined abnormalities. To specifically analyze the quantitative CA cutoff values and the correlation of clone size to survival outcomes, we established subgroupings with positivity defined as 1q21+ or 1q23+ present in more than 20% of cells and t(4;14) present in more than 30% of cells. Patients who satisfied both cutoff criteria were labeled as 'double-hit', and patients with CA present, but clone sizes less than the prespecified cutoffs for both CA, were denoted as 'CA(low)'. All tests were two-sided with p-values <0.05 considered statistically significant. Results: The median age at diagnosis was 61.5 years, 54% were male, and 84% of patients identified as non-Hispanic White. Of the total cohort and independent of quantitative CA cutoff, 1q+ (1q21+ or 1q23+) only was the most prevalent CA in 198 (81%) of patients, t(4;14) only in 13 (5%) patients, and both 1q+ (1q21+ or 1q23+) and t(4;14) in 32 (13%) patients. One hundred seventy (70%) patients underwent ASCT, followed by maintenance therapy in 85% of them. Pre-transplant best hematologic responses did not differ amongst CA groups. There was no difference in baseline hemoglobin, serum creatinine, calcium, albumin, lactate dehydrogenase, or beta-2 microglobulin amongst the CA groups. No differences in outcomes were noted without including size clone cutoffs. There was a statistically significant difference in univariable analysis in PFS and OS from ASCT in patients with 1q21+ and t(4;14) 'CA(low)’ (n=76, 31%) having better survival outcomes compared to the corresponding 'double-hit’ group (n=25, 10%) (PFS: HR=0.52, 95% CI: 0.30-0.92, p=0.025; OS: HR=0.41, 95% CI: 0.21-0.81, p=0.010). This pattern in PFS and OS after ASCT was also seen in patients with 1q23+ and t(4;14) 'CA(low)’ (n=119, 49%) faring better compared to its 'double-hit’ group (n=19, 8%) (PFS: HR=0.47, 95% CI:0.26-0.83, p=0.009; OS: HR=0.34, 95% CI:0.18-0.66, p=0.001). Patients with t(4;14) only (>30%) (n=19, 8%) within the 1q23+ cohort also demonstrated longer PFS and OS after ASCT as compared to its 'double-hit’ group (PFS: HR=0.42, 95% CI:0.20-0.91, p=0.027; OS: HR=0.38, 95% CI:0.15-0.94, p=0.037). After adjusting for age and R-ISS staging, the difference between 1q23+ and t(4;14) 'CA(low)’ vs 'double-hit’ and t(4;14) only within the 1q23+ cohort vs 'double-hit’ remained significant in multivariable analysis in PFS after ASCT ('CA(low)’ group: HR=0.50, 95% CI:0.25-0.99, p=0.048; and t(4;14) only group: HR=0.41, 95% CI:0.18-0.98, p=0.045). Conclusion: Our findings reveal the differences seen in MM patients with single CA compared to the combined high-risk aberrations with both 1q21+/1q23+ and t(4;14). The effects of quantitative clone sizes on survival outcomes were displayed as patients with smaller clone sizes (i.e. 'CA(low)’ subsets) had superior survival outcomes than those patients with 'double-hit’ 1q21+ (or 1q23+) >20% and t(4;14) >30%. It is thus imperative for clinicians to be mindful of not only the presence of high-risk CA but also the size of the aberrant clone influencing subsequent MM disease characteristics.

PTPN1 Deficiency Accelerates the Progression to Myelofibrosis in JAK2V617F-Driven Myeloproliferative Neoplasm

The deletion of chromosome 20q (del20q) is frequently associated with myeloid malignancies including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The del20q cytogenetic abnormality was observed most frequently in patients with myelofibrosis (MF). However, the key target genes within 20q involved in the pathogenesis of myelofibrosis has remained elusive. We previously reported that PTPN1 gene encoding protein tyrosine phosphatase non-receptor type 1 is a potential tumor suppressor within 20q in MPN/MF (Jobe et al. Leukemia 2017). The del20q involving PTPN1 deletion was more commonly found in MPN/MF in comparison to other myeloid neoplasms. We also observed significant co-occurrence of del20q involving PTPN1 deletion with JAK2V617F mutation in patients with MF. So, we hypothesize that PTPN1 deficiency may cooperate with JAK2V617F in the pathogenesis of MF. In this study, we investigated the effects of heterozygous and homozygous PTPN1 deletion in JAK2V617F-induced MPN using a conditional PTPN1 knockout allele and a JAK2V617F knock-in mouse model of MPN. We previously reported the generation of a conditional JAK2V617F knock-in mouse. Mice expressing heterozygous JAK2V617F (JAK2VF/+) exhibit a PV-like MPN characterized by increase in red blood cells (RBC), white blood cells (WBC), neutrophils and platelets in the peripheral blood. Heterozygous deletion of PTPN1 significantly increased WBC, neutrophil and platelet counts but reduced RBC and hemoglobin levels in JAK2VF/+ mice. Flow cytometric analysis showed that heterozygous deletion of PTPN1 significantly increased myeloid (Gr-1+Mac-1+) and megakaryocytic (CD41+CD61+) precursors in the BM and spleens of PTPN1-deficient JAK2VF/+ mice compared with JAK2VF/+ mice. Additionally, deletion of PTPN1 significantly increased hematopoietic stem cells and myeloid progenitors in the BM and spleens of PTPN1-deficient JAK2VF/+ mice compared to JAK2VF/+ mice. Spleen weight was significantly increased in JAK2VF/+ mice, while deletion of PTPN1 further enhanced splenomegaly in PTPN1-deficient JAK2VF/+ mice. Furthermore, deletion of PTPN1 significantly enhanced bone marrow fibrosis in JAK2VF/+mice. Homozygous deletion of PTPN1 resulted in more robust and accelerated development of lethal myelofibrosis in JAK2VF/+ mice. The cooperative effects of JAK2V617F and PTPN1 deficiency in myelofibrotic transformation is associated with increased production of proinflammatory cytokines including IL-1beta, IL-6 and TGF-beta. We also observed enhanced activation of STAT1, STAT3, ERK1/2 and NF-kB in PTPN1-deficient JAK2VF/+ mice bone marrow compared with JAK2VF/+ or WT mice bone marrow. CRISPR/Cas9-mediated deletion of PTPN1 also resulted in enhanced activation of these signaling molecules and increased proliferation in JAK2V617F-positive mouse and human hematopoietic cells. Furthermore, deletion of PTPN1 significantly reduced ruxolitinib sensitivity against JAK2V617F-positive hematopoietic cells, suggesting that del20q involving PTPN1 deletion may cause ruxolitinib resistance in MPN. Overall, this study demonstrates that PTPN1 is an important target in del20q-associated MPN, and deficiency of PTPN1 collaborates with JAK2V617F in the progression to myelofibrosis.

EZH2 Inhibition Mediates Proptosis Via DHX9 and S100A9 in Myelodysplastic Syndromes

Myelodysplastic Syndromes (MDS) is considered as an archetypal epigenetic disorder due to the aberrant epigenome, frequent epigenetic regulator mutations and the most responsive disease to DNA methylation inhibitors. Among numerous epigenetic regulators, Enhancer of zeste 2 homologue 2 (EZH2) occupies an important position owing to its involvement in multiple epigenetic modifications. In MDS, EZH2 mutation frequency is 5%-10%. Given that the EZH2 gene is located at 7q36.1, abnormalities of chromosome 7q disturb EZH2 as well. To investigate the expression and effects of EZH2 in MDS patients without EZH2 mutation and chromosome 7 abnormalities, qPCR was performed. We found that EZH2 transcript level was significantly higher in MDS patients and its expression was highest in very high risk group, followed by the high risk group. The survival time of EZH2 high expression was shorter than the low expression group. Multivariable Cox regression showed that EZH2 expression was an independent prognostic factor. Next, we treated MDS cell lines MDS-L and SKM-1 with EZH2 inhibitor DZNep and found that DZNep inhibited cell proliferation in a dose- and time-dependent manner. To determine which pattern of cell death played the most important role, we pretreated different cell death inhibitors for 1 hour before adding DZNep. Compared with the group without cell death inhibitors, pyroptosis inhibitor (VX765) was the most effective in restoring cell viability. Besides, both DZNep and knockdown of EZH2 could trigger pyroptosis. To elucidate the possible mechanism of pyroptosis induced by EZH2 inhibition, we searched for RNA-seq data from tumor cells treated with EZH2 inhibitors by using the GEO database. We overlapped 53 genes by analyzing GSE151576, GSE80239 and GSE119088, of which DHX9 was the highest expressed in BM and attracted attention.Meanwhile, down-regulation of DHX9 was observed after pharmacological inhibition or EZH2 knockdown. There was a positive correlation between EZH2 and DHX9 in MDS patients. Furthermore, DHX9 expression was increased in MDS patients in contrast to control, and its overexpression was connected with shorter OS. Knockdown of DHX9 with two shRNAs induced pyroptosis. CUT&RUN and qPCR assay results validated that EZH2 and H3K4me3 occupancy at DHX9 promoter. Moreover, with RNA sequencing we found DHX9 can regulate S100A9 and confirmed by western blot. Overexpression of S100A9 could cause pyroptosis of MDS cell lines, which could be reversed by a S100A9 inhibitor. DHX9 and S100A9 are negatively correlated in MDS patients. Finally, we established xenograft mouse models by intravenously injecting luciferase-labeled SKM-1 cells. Intraperitoneal injection of 2mg/kg DZNep daily could significantly reduce the tumor burden and prolong the survival time of mice. We further confirmed that inhibition of EZH2 regulated DHX9 and S100A9 and induced pyroptosis in mouse bone marrow cells. In conclusion, we found that EZH2 expression in de novo MDS patients without EZH2 mutation and chromosome 7 abnormality was higher than that of the control group, and its high expression was correlated with inferior prognosis. EZH2 suppression inhibited tumor growth and induced pyroptosis both in vitro and in vivo. Inhibition of EZH2 triggered pyroptosis by reducing its binding in the DHX9 promoter and upregulating S100A9. In this present study, we reveal a novel molecular mechanism by which EZH2 inhibition induced pyroptosis and provide a basis for the treatment of MDS with EZH2 inhibitor. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Therapy-Related Myelodysplastic Syndromes with Isolated Del(5q): Comparative Analysis of Phenotype and Long-Term Survival

Background: Myelodysplastic syndromes (MDS) with chromosome 5q deletion [MDS-del(5q)] is a subcategory of MDS recognized by both the International Consensus (ICC) and World Health Organization (WHO) classification systems. Diagnostic criteria according to ICC-2022 include i) presence of del(5q) alone or with only one other additional cytogenetic abnormality, other than -7/del(7q), ii) absence of multi-hit TP53 mutation, iii) presence of ≥1 cytopenias, iv) presence of bone marrow myeloid dysplasia in ≥1 lineages, v) absence of ≥5% bone marrow blasts, and vi) absence of ≥2% circulating blasts (Arber et al. Blood 2022). We used WHO-2016 diagnostic criteria to identify Mayo Clinic patients with MDS-del(5q) and confirmed a study population that also met ICC-2022 criteria. The main objective of the current study was to describe the phenotype and long-term survival of patients with therapy-related MDS-del(5q) and compare the findings with a cohort of patients with primary MDS-del(5q), sourced from the same database. Methods: The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from patients with MDS-del(5q) who were seen at the Mayo Clinic. Diagnosis of MDS-del(5q) was retrofitted to comply with ICC-2022 criteria, with the exception that patients with additional cytogenetic abnormalities were excluded from the current report (Arber et al. Blood 2022). Most patients were treated with lenalidomide, often given at the recommended dose of 10 mg/day x 21-28 days, with dose adjustements at physician discretion. Erythroid response criteria included transfusion independence for at least 8 weeks for transfusion dependent patients and an increase in hemoglobin by 1.5 g/dl lasting for at least 8 weeks, in transfusion independent patients. Transfusion dependency was assigned to those patients who had received at least 3 units of packed red cells in the 8 weeks prior to diagnosis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results A total of 93 patients (median age 73 years; range 24-89; females 63%) were included in the current study, including primary (n=79) and therapy-related (n=14) MDS-del(5q). Cancer/treatment history for the latter group of patients included prostate cancer/radiotherapy (n=5), thyroid cancer/radiotherapy (n=2), breast cancer/radiotherapy (n=2), bladder cancer/chemotherapy (n=2), colon cancer/chemotherapy (n=1), lung cancer/chemotherapy (n=1), and non-Hodgkin's lymphoma/chemotherapy/radiotherapy (n=1). Median (range) time from radiotherapy/chemotherapy and recognition of MDS-del(5q) was 16 years (1.9-30). Comparison of presenting features for therapy-related vs primary MDS-del(5q) revealed the former to be associated with older age (median 77 vs 72 years; p=0.002), male gender (72% vs 30%; p=0.003), and lesser likelihood of transfusion-dependent anemia, at time of initial diagnosis (28% vs 68%; p=0.03). The two groups were otherwise similar in their leukocyte and platelet counts and mutational landscape, including prevalence of TP53 mutations (25% vs 13%, respectively). All 79 patients with primary vs 10 of the 14 with therapy-related disease received lenalidomide therapy with erythroid response rates of 62% vs 80% (p=0.2) and complete/partial cytogenetic response rates of 13%/10% vs 0/0, respectively (p=0.05); the latter information was available in a subset of 64 patients. After a median follow up of 4.8 years, 38 (41%) deaths and 16 (17%) leukemic transformations were documented; the corresponding figures for therapy related/primary disease were 50%/39% and 14%/18%, respectively (p=0.4 and 0.7). Figure 1 depicts similar (p=0.6) survival data for therapy-related vs primary MDS-del(5q), with corresponding median survival (3-year survival) rates of 6.3 years (60%) vs 7.7 years (63%). Conclusions: The current study describes the circumstances surrounding the development of therapy-related MDS-del(5q) and highlights differences in age and gender distribution, compared to primary MDS-del(5q). The two groups were otherwise similar in their response to lenalidomide therapy, overall survival, and leukemic transformation rates while their similarity in mutation distribution requires validation with higher number of informative cases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TP53 Mutation Status Defines a Distinct Clinicopathological Entity of Therapy-Related Myeloid Neoplasm, Characterized By Genomic Instability and Extremely Poor Outcome

BACKGROUND: The prognostic and therapeutic impact of TP53-mutated (TP53mut) myeloid neoplasm (MN) is an area of intense interest, with the 5th edition of the World Health Organization, the International Consensus Classification, and the European LeukemiaNet guidelines, all recommending major changes MN classification based on TP53mut. However, studies driving these changes predominantly included de novo MDS (Bernard et al. Nat Med 2020), AML and high risk MDS treated with intensive chemotherapy (Grob et al. Blood 2022), or MN with complex karyotype (CK, Weinberg et al. Blood Adv 2022). Therefore, the significance of these findings as they relate to t-MN is not known. METHODS: We analyzed therapy-related myeloid neoplasm (t-MN) (n=377) patients evaluated at Mayo Clinic, Rochester and South Australian MDS Registry with clinical, integrated genomic profiling, and long-term follow-up to characterize the genomic profile and outcome of TP53mut t-MN. RESULTS: Our cohort of 377 t-MN included t-MDS (n=245, 65%) and t-AML (n=132, 35%), with a median age at t-MN diagnosis being 65.7 years. 184 TP53mut at variant allele frequency (VAF) ≥2% were detected in 131 (34.74%) patients. Median overall survival (OS) of TP53mut VAF <10% (n=14) was similar to wild-type TP53 (TP53wt), hence further analysis was restricted to TP53mut with VAF >10% (n=111). TP53mut patients without VAF (n=6) were not included for further analysis. Genomic instability was highly evident in TP53mut t-MN compared to TP53wt t-MN. CK, monosomal karyotype (MK), chromosome 5q deletion, and marker chromosomes were enriched in TP53mut t-MN. In contrast, driver oncogenic gene mutations such as IDH2, SRSF2, RAS, and TET2 were less frequent in TP53mut. TP53mut also dictated the clinical presentation, longer latency (interval between primary cancer and t-MN diagnosis), and inferior OS compared to TP53wt (8.2 vs. 19.4 months; P <0.001). On multivariate Cox regression analyses, TP53mut was associated with inferior OS (HR 1.43 95% CI 1.01-2; P =0.03) independent of CK and bone marrow blast %. A significantly higher proportion of CK patients had TP53mut compared to non-CK patients (74.4% vs. 5.4%, P <0.001). Even within the CK cohort, TP53mut was associated with structural genomic instability, with a higher proportion of TP53mut harbouring MK (P =0.013), marker chromosome (P =0.033), deletion 5q (P <0.001), abnormal chromosome 17 (P =0.001), and other structural chromosomal aberrancies compared to TP53wt-CK. In contrast to the predominance of structural chromosomal aberrancies in the TP53mut-CK cohort, mutations driving MN were sparse in TP53mut-CK compared to TP53wt-CK. Finally, TP53mut-CK was associated with significantly poor survival compared to the TP53wt-CK (8.2 vs. 17 months; P <0.001). Though bone marrow (BM) % predicted poor outcome in the whole t-MN cohort, it failed to predict outcome in TP53mut t-MN patients. We compared the clinical and molecular profiling of TP53mut t-MDS (<5%, 5-9%, 10-19% blasts) and t-AML (≥20% blasts) at t-MN diagnosis. Genomic instability, TP53mut allelic status, number of TP53mut, and the clone size were similar across all four cohorts. Remarkably, the survival of TP53mut patients were equally dismal irrespective of blast percentage (P=0.2, Figure 1A). Together, these findings suggest that TP53mut t-MN represent a homogenous group irrespective of BM blast percentage. Integrating the mutation, conventional cytogenetic, copy number variation, FISH, and SNP array analysis, TP53mut were classified as single- or multi-hit (Grob et al. Blood 2022). In total, 31 (25.2%) of the 123 patients with TP53mut (n=111) and/or loss of TP53 (n=12) locus were considered single-hit, and 92 (74.8%) were considered as multi-hit. In contrast to prior reports, we did not observe enrichment of genomic instability, or differences in degree of cytopenia, bone marrow blast % and t-MN phenotype in multi- vs single-hit TP53mut patients. Importantly, no OS differences were observed when TP53mut were stratified by type of TP53mut (truncation vs. missense), site of missense mutation (hot- vs. non-hot spot) and single- vs multi-hit (Figure 1B). CONCLUSION: The classification of t-MN based on TP53mut status is clinically and biological relevant, irrespective of blast % or single- or multiple-hit status. TP53mut t-MN is a molecularly defined unique group enriched for genetic instability and is associated with extremely poor outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

NOTCH1 Signaling Is Dysregulated in CLL By the Deubiquitinase USP28 That Is Recurrently Affected By Del(11q) and Can be Therapeutically Targeted

Active NOTCH1 signaling is present in approximately half of chronic lymphocytic leukemia (CLL) patients and was shown to be a marker for disease outcome. The NOTCH1 signaling pathway can be aberrantly activated through mutations in the NOTCH1 gene itself or co-factors such as FBXW7, resulting in protein stabilization of cleaved NOTCH1 (NICD). However, 50% of CLL cases with NOTCH1 activation do not harbor alterations in these genes, suggesting additional mechanisms for NOTCH1 hyperactivation. Since NICD can be stabilized due to defects in the ubiquitination/degradation machinery, we aimed to elucidate the role of the on chromosome 11q23 located deubiquitinase USP28 in the regulation of NOTCH1 activity and as a novel therapeutic target for NOTCH1-dependent CLL patients. First, we evaluated USP28 expression levels in a previously published cohort of 285 untreated CLL patients characterized via SNP array analysis and gene expression profiling (Edelmann et al, Blood 2012; Bloehdorn et al, Nat Commun 2021). The presence of monoallelic USP28 deletion was identified in 90% of CLL patients harboring a heterozygous deletion of chromosome 11q (del(11q); n=96), resulting in a significant decrease of USP28 RNA expression in comparison to non-del(11q) cases (n=199, P<0.0001). In non-del(11q) cases, USP28 expression levels were variable and assessment of protein levels of USP28 and its target proteins by western blotting in primary CLL cells (n=8) revealed a correlation between high protein levels of USP28 and remarkably high levels of NICD and other USP28 targets such as c-MYC or c-JUN. In addition, interaction of USP28 with NICD was shown by immunoprecipitation assays. In parallel, we implemented the CRISPR/Cas9 system to generate HG3 CLL-derived cell lines harboring USP28 biallelic deletion (HG3-USP28KO, n=5), monoallelic 11q deletion (HG3-del(11q), 17 Mb size including both ATM and USP28 genes, n=3), biallelic ATM truncation (HG3-ATMKO, n=3) or the combination of both events (HG3-del(11q) ATMKO, n=3). Overexpression of USP28 in HG3WT and HG3-USP28KO cells increased NOTCH1 activity measured by luciferase reporter assays (P<0.05, P<0.001, respectively). In contrast, in HG3-del(11q) cells decreased USP28 RNA and protein expression resulted in reduced NOTCH1 activity compared to HG3WT cells (P<0.05). Notably, HG3-del(11q) ATMKO cell lines showed even lower levels of NOTCH1 activity (P<0.01). Given that ATM phosphorylates USP28 (Zhang et al, Cell 2006), we hypothesized that in CLL cells ATM regulates USP28 activity and thereby also downstream NOTCH1 signaling. Indeed, NOTCH1 activity was reduced in HG3-ATMKO cells (P<0.01). However, USP28 overexpression in HG3-ATMKO cells did not lead to increased NOTCH1 signaling activity as observed in HG3WT cells. Moreover, pharmacological ATM inhibition (5 µM of KU-55933) also resulted in reduced NOTCH1 activity in HG3WT cells (P<0.0001) and reduced NICD levels in primary CLL cells (n=12; P<0.05). We finally explored the impact of the small molecule USP28 inhibitor AZ1 (alone or in combination with ibrutinib or venetoclax) on NICD protein levels and viability of primary CLL cells (n=15). AZ1 treatment (10 µM) resulted in decreased NICD levels, independent of mutations within NOTCH1 or FBXW7 (P<0.01). ATP quantification and 7-AAD staining revealed that increasing doses of AZ1 significantly reduced viability of NOTCH1/FBXW7-MUT primary cells (n=5) as well as of NOTCH1/FBXW7-WT cells (n=6) expressing high protein levels of USP28 and NICD (P<0.01; P<0.05, respectively). In contrast, AZ1 treatment did not affect viability of CLL patient cells expressing low NICD levels (n=4; P=0.56) Furthermore, AZ1 combined with venetoclax potentiated the reduction of cell viability especially in NOTCH1/FBXW7-MUT or patient cells expressing high USP28 and NICD levels (P<0.001; P<0.05). We propose that high levels of the deubiquitinase USP28 are responsible for hyperactivation of NOTCH1 signaling in CLL. This hypothesis is further confirmed in del(11q) CLL cells, where USP28 heterozygous deletion resulted in low NOTCH1 activity. In addition, our data reveal that USP28 activity is regulated by ATM, uncovering a novel mechanism of NOTCH1 modulation via the ATM-USP28-NOTCH1 axis (Figure 1). Finally, we demonstrate that USP28 inhibition by AZ1 suppresses USP28-mediated NOTCH1 activation and could represent a novel therapeutic strategy in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CSIG-22. THE MOLECULAR NETWORK OF CAMTA-1 IN GLIOMA CELLS

Abstract Calmodulin-binding transcription activator 1 (CAMTA1) is a transcription factor located on chromosome 1p, and we have recently reported that CAMTA1 expression reduced neurosphere formation and tumor growth in nude mice, suggesting that CAMTA1 can function as tumor suppressor in experimental glioma. In the present study, we investigated the molecular network of CAMTA-1 in glioma cells. We established glioma cell lines with a doxycycline-inducible overexpression of CAMTA1. We performed a cap analysis of gene expression (CAGE) on these overexpressing cell lines to identify differentially regulated transcriptional start sites. We particularly focused on regulated target genes encoded on chromosome 19q. We identified an interaction of CAMTA1 overexpression with changes in the differential expression of Zink finger (ZNF) genes located on 19q. We will present novel data on a potential molecular interaction between chromosomes 1p and 19q.

PATH-11. THE LONGITUDINAL EVOLUTIONARY TRAJECTORY OF OLIGODENDROGLIOMA IS DRIVEN BY TREATMENT-ASSOCIATED GENETIC ALTERATIONS

Abstract Oligodendroglioma is a subtype of diffuse glioma defined by a mutation in the isocitrate dehydrogenase (IDH) genes and a co-deletion of chromosome arms 1p and 19q. These tumors primarily occur in adult patients in their third and fourth decade of life and are universally fatal due to an inevitable recurrence that follows a treatment regimen of surgical resection and an optional combination of alkylating chemotherapy and/or radiation therapy. While initially slow growing, recurrent tumors exhibit increasingly aggressive phenotypes that become progressively more difficult to treat with conventional therapy. Currently, the molecular mechanisms and cellular phenotypes that drive this recurrence remain unknown. To understand these factors, we assembled a cohort of matched initial and recurrent oligodendroglioma samples from over 100 patients and performed whole-genome sequencing and whole-exome sequencing on each of them. To link these molecular profiles to cell state changes, we additionally performed bulk and single-nucleus RNA-sequencing on a subset of these tumor pairs. In nearly 40% of alkylating chemotherapy-treated patients, recurrent tumors presented with hypermutation that corresponded with an increase in neoplastic cell proliferation. Additionally, while individual somatic alterations specific to recurrence were relatively rare, we observed a subset of tumors that acquired deletions in the cell cycle regulator CDKN2A following treatment with radiotherapy. Acquisition of either of these features associated with shorter patient survival and higher grade at recurrence, implicating cell cycle dysregulation as a mechanism of treatment resistance and increased tumor severity. Together, these results indicate that oligodendrogliomas evolve in a treatment-specific manner following chemo- and radiation therapy and highlight key pathways that can be targeted to delay the onset of recurrence.

Sensory ataxic neuropathy with dysarthria and ophthalmoparesis (SANDO) syndrome associated with two heterozygous POLG mutations

Polymerase gamma is a mitochondrial DNA polymerase, that is responsible for the replication of the mitochondrial DNA (mtDNA). It is encoded by the POLG gene, on chromosome 15q25. Various mutations in this gene have been described, with varied phenotypic manifestations. The triad of sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO) has been reported only in a small group of patients with POLG mutations. We report the case of a male, who presented with phenotype of SANDO syndrome and and was found to have two pathogenic, heterozygous mutations in the POLG gene.