First person – Ajay Labade and Adwait Salvi

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Ajay Labade and Adwait Salvi are co-first authors on ‘ Nup93 and CTCF modulate spatiotemporal dynamics and function of the HOXA gene locus during differentiation’, published in JCS. Ajay is a postdoctoral researcher in the lab of Dr Jason Buenrostro at Harvard University, Cambridge, USA, where he develops multimodal genomics technologies for the investigation of functional genome organization. Adwait is a PhD student in the lab of Filippo Rijli at Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland, who works on deciphering the role of 3D genome organization in gene regulation during differentiation.

Nup93 and CTCF modulate spatiotemporal dynamics and function of the HOXA gene locus during differentiation

Nucleoporins regulate nuclear transport and are also involved in DNA damage, repair, cell cycle, chromatin organization, and gene expression. Here, we studied the role of nucleoporin Nup93 and the chromatin organizer CTCF in regulating HOXA expression during differentiation. ChIP sequencing revealed a significant overlap between Nup93 and CTCF peaks. Interestingly, Nup93 and CTCF are associated with the 3' and 5′HOXA genes respectively. Depletions of Nup93 and CTCF antagonistically modulate expression levels of 3′and 5′HOXA genes in undifferentiated NT2/D1 cells. Nup93 also regulates the localization of the HOXA gene locus, which disengages from the nuclear periphery upon Nup93 but not CTCF depletion, consistent with its upregulation. The dynamic association of Nup93 and CTCF with the HOXA locus during differentiation correlates with its spatial positioning and expression. While Nup93 tethers the HOXA locus to the nuclear periphery, CTCF potentially regulates looping of the HOXA gene cluster in a temporal manner. In summary, Nup93 and CTCF complement one another in modulating the spatiotemporal dynamics and function of the HOXA gene locus during differentiation.

Case Study: Patient with 7p14–P21 Deletion Spanning the TWIST Gene and the HOXA Gene Cluster

Introduction: While several literature reports have been published about patients with microdeletions within chromosome 7p, only a small fraction of those reports is specific to deletions that encompass the TWIST gene and HOXA gene cluster. The large-span deletions within this cluster result in haploinsufficiency of six genes known to have a role in different autosomal dominant genetic disorders: TWIST1, GSDME (DFNA5), CYCS, HOXA11, HOXA13, and GARS. Deletion of TWIST1 gene on 7p21 and deletion of HOXA cluster on 7pl5.2 lead to Saethre-Chotzen syndrome and to hand-foot-genital syndrome, respectively. Objectives: Our patient presented with a phenotype combining Saethre-Chotzen syndrome (SCS) and hand-foot-genital syndrome (HFS), which is similar to previously reported cases with a deletion spanning 7p21– p14.3. The objective of our report is to correlate the clinical observations with the patient’s genetic test result, namely 46,XY,del(7)(p14p21). Patient and Methods: We describe a patient who had manifestations of SCS and HFU, caused by an interstitial deletion of chromosome 7p21–p14 detected by RHG band. Results and Conclusion: We therefore confirm previous reports that microdeletions of 7p spanning the TWIST gene and HOXA gene cluster lead to a clinically recognizable ‘haploinsufficiency syndrome’. All of the features of this patient could be accounted for by combined effect of the deletion of the TWIST and HOXA cluster.

NUTM1-Rearranged Infant and Pediatric B Cell Precursor Acute Lymphoblastic Leukemia: A Good Prognostic Subtype Identified in a Collaborative International Study

Background and Aims A novel genetic subtype of B cell precursor acute lymphoblastic leukemia (B-ALL) is characterized by rearrangement of NUTM1 (NUTM1r) on 15q14 resulting in fusion of NUTM1 to one of several partner genes such as CUX1, ACIN1, BRD9, and IKZF1. The downstream effects of NUTM1r include upregulation of the proto-oncogene BMI1 and specific fusions also induce transcription of the HOXA gene cluster (Hormann et al. Haematologica 2019; Li et al. PNAS 2018). This novel subtype is rare in children, but appears to be more prevalent among infants negative for KMT2A rearrangement (KMT2Ar) based on the frequency of karyotypic 15q aberrations (De Lorenzo et al. Leukemia 2014). This international collaborative study aimed to determine the frequency of NUTM1r in infant and pediatric cohorts, and to characterize the demographic, clinical and molecular features of NUTM1r-positive B-ALL. Patients and Methods Interfant-related study groups provided NUTM1 screening results for KMT2Ar-negative Interfant-99 and -06 cases with karyotypic 15q aberration, normal karyotype, or missing karyotype. Additionally, NUTM1r-positive cases of any age were collected from the study groups united in the Ponte di Legno consortium. The identified NUTM1r-positive children were diagnosed between 1995-2019, infants (≤365 days of age) included in the Interfant-99 or 06 trials were diagnosed between 2000-2016, and remaining infants between 1986-2019. The techniques used for the detection of NUTM1r were break-apart FISH, RNA sequencing, and RT-PCR. Event-free survival (EFS) and overall survival (OS) were estimated according to Kaplan-Meier, standard error according to Greenwood, and the curves were compared by log-rank test. Results We identified 81 NUTM1r cases, including 35 Interfant-enrolled infants, 10 other infants and 36 children. NUTM1r was reported to be rare among pediatric B-ALL with an estimated frequency range of 0.28-0.86%. The median age among NUTM1r-positive children was 4.5 years (range 1-15). Among KMT2Ar-negative infants the frequency of NUTM1r was 21.7%. Of NUTM1r-positive infants, 54% were <6 months at diagnosis (median 5.6, range 0.4-11.0 months) compared with 16% in the remaining KMT2Ar-negative infants (median 9.3, range 0.1-11.9; p<0.0001). Other baseline characteristics (WBC, gender, prednisone poor response) were similar between NUTM1r-positive and -negative infants. Of the NUTM1r-positive cases, all achieved complete remission, 82% had minimal residual disease <10e-4 at the end of induction, and no patient received stem cell transplant in first remission. The 4-year EFS was 100% in Interfant-enrolled NUTM1r-positive patients versus 74% (95% CI 65.1-81.0, p=0.001) in the remaining KMT2Ar-negative cases (n=126). The better outcome was confirmed also after adjusting for WBC, gender and prednisone response (p=0.0001). The 4-year OS were 100% and 88.0% (95% CI 80.5-92.7) for NUTM1r-positive and other KMT2Ar-negative infant cases, respectively (p-value=0.04). Children and non-Interfant-enrolled infants treated on different treatment protocols showed 89.4% (95% CI 78.6-1) 4-year EFS and 100% 4-year OS. In order of frequency, NUTM1 fusion partners were ACIN1 (30.4%), CUX1 (21.7%), BRD9 (17.4%), ZNF618 (13%), AFF1 (4.3%), SLC12A6 (4.3%), IKZF1 (2.9%), and three novel partners: ATAD5 (2.9%), CHD4 (1.4%) and RUNX1 (1.4%). Infants mainly showed fusions with ACIN1, CUX1, BRD9 and AFF1, associated with HOXA9 upregulation. Older infants and children showed both HOXA-upregulating and non-HOXA-upregulating fusions. Epigenetic profiling showed a distinct pattern of DNA methylation and histone modification of the HOXA gene cluster region in leukemic cells of an ACIN1-NUTM1 pediatric case compared with KMT2Ar-positive and KMT2Ar/NUTM1r-negative pediatric cases. Conclusions NUTM1r ALL was identified as the second largest subtype in infants, found in 21.7% of KMT2Ar-negative infant B-ALL, representing 5-7% of total infant ALL, and associated with excellent outcome on Interfant standard risk protocols. The favorable outcome was confirmed in the Ponte di Legno cohort of infant and pediatric NUTM1r-positive patients enrolled on different treatment protocols over more than two decades. We conclude that NUTM1r ALL is a favorable genetic subtype in infants and children and possibly eligible for treatment reduction. Disclosures No relevant conflicts of interest to declare.

Landscape Of HOXA Genes Methylation in Colorectal Cancer

Colorectal cancer (CRC) is among the most common cancers worldwide and the second leading cause of cancer-related death in Malaysia. The HOXA gene cluster is a family of Homeobox A genes encoding transcriptional regulators that play vital roles in cancer susceptibility and progression. Dysregulated HOXA expression influences various aspects of carcinogenesis processes. Therefore, this study aims to elucidate the methylation landscape of HOXA genes in CRC. Twelve pairs of CRC — adjacent normal tissues were subjected to Infinium DNA MethyEPIC array. Differentially methylatedregions were identified using the ChAMP Bioconductor and methylation levels of HOXA genes were manually curated. We identified 100 significantly differentially methylated probes annotated to HOXA genes. HOXA3 has the highest number of differentially methylated probes (n=27), followed by HOXA2 (n=20) and HOXA4 (n=14). The majority (43%) of the probes were located at the transcription start site (TSS) 200, which is one of the gene promoters. In respect to CpG islands (CGI), the probes were equally located in the island and shore regions (47% each) while a minor percentage was in the shelf (6%). Our work gave a comprehensive assessment of the DNA methylation pattern of HOXA genes and provide the first evidence of HOXA2, HOXA3 and HOXA4 differential methylation in Malaysian CRC. The new knowledge from this study can be utilized to further increase our understanding of CRC methylomics, particularly on the homeobox A genes. The prognostic and diagnostic roles of the differentially methylated HOXA genes warrant future investigations.