Search

US-12624398-B2 - Use of long non-coding RNAs in medulloblastoma

US12624398B2US 12624398 B2US12624398 B2US 12624398B2US-12624398-B2

Abstract

The present invention relates to the field of cancer. More specifically, the present invention provides compositions and methods useful for detecting long non-coding RNAs (lncRNA) in medulloblastoma. In one embodiment, the present invention provides a method comprising detecting lnc RNA HLX2-7 in a biological sample obtained from a patient having or suspected of having medulloblastoma. In certain embodiments, detecting step is performed using RNA fluorescence in situ hybridization (FISH) assay. In specific embodiments, the biological sample is a tissue sample. In particular embodiments, the tissue sample is a formalin-fixed paraffin-embedded (FFPE) sample. In a specific embodiment, the FISH assay comprises oligonucleotide probes that hybridize to lncHLX2-7 (SEQ ID NO:200) and branched DNA signal amplification.

Inventors

  • Joseph Ranjan Perera
  • Keisuke KATSUSHIMA

Assignees

  • THE JOHNS HOPKINS UNIVERSITY

Dates

Publication Date
20260512
Application Date
20200914

Claims (20)

  1. 1 . A method comprising detecting long non-coding (lnc) RNA HLX2-7 in a biological sample obtained from a patient having or suspected of having medulloblastoma, wherein the detecting step is performed using RNA fluorescence in situ hybridization (FISH) assay.
  2. 2 . The method of claim 1 , wherein the biological sample is a tissue sample.
  3. 3 . The method of claim 2 , wherein the tissue sample is a formalin-fixed paraffin-embedded (FFPE) sample.
  4. 4 . The method of claim 1 , wherein the FISH assay comprises oligonucleotide probes that hybridize to lncHLX2-7 and branched DNA signal amplification.
  5. 5 . The method of claim 4 , wherein the probes comprise at least one of SEQ ID NOS: 3-4 and 8-21.
  6. 6 . The method of claim 4 , wherein the probes comprise SEQ ID NOS: 2-3 and 8-21.
  7. 7 . The method of claim 5 , wherein the probes further comprise at least one of SEQ ID NOS: 5-7.
  8. 8 . The method of claim 6 , wherein the probes further comprise SEQ ID NOS: 5-7.
  9. 9 . The method of claim 1 , further comprising detecting MYC expression in the biological sample.
  10. 10 . The method of claim 9 , wherein the biological sample is a tissue sample.
  11. 11 . The method of claim 10 , wherein the tissue sample is a formalin-fixed paraffin-embedded (FFPE) sample.
  12. 12 . The method of claim 9 , wherein the FISH assay comprises oligonucleotide probes that hybridize to MYC and branched DNA signal amplification.
  13. 13 . The method of claim 12 , wherein the probes comprise at least one of SEQ ID NOS: 51-56, 59-60, 62-63, 66-69, 72-73, 75-78, 81-98, 101-102.
  14. 14 . The method of claim 13 , wherein the probes comprise SEQ ID NOS: 51-56, 59-60, 62-63, 66-69, 72-73, 75-78, 81-98, 101-102.
  15. 15 . The method of claim 13 , wherein the probes further comprise at least one of SEQ ID NOS: 57-58, 61, 64-65, 70-71, 74, 79-80, 99-100.
  16. 16 . The method of claim 14 , wherein the probes further comprise SEQ ID NOS: 57-58, 61, 64-65, 70-71, 74, 79-80, 99-100.
  17. 17 . The method of claim 9 , further comprising detecting Inc RNA SPRY4-IT1 in the biological sample.
  18. 18 . The method of claim 17 , wherein the biological sample is a tissue sample.
  19. 19 . The method of claim 18 , wherein the tissue sample is a formalin-fixed paraffin-embedded (FFPE) sample.
  20. 20 . The method of claim 17 , wherein the FISH assay comprises oligonucleotide probes that hybridize to Inc SPRY4-IT1 and branched DNA signal amplification.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a 35 U.S.C. § 371 U.S. national entry of International Application PCT/US2020/050679, having an international filing date of Sep. 14, 2020, which claims the benefit of U.S. Provisional Application No. 62/938,526, filed Nov. 21, 2019, and U.S. Provisional Application 62/899,619, filed Sep. 12, 2019, each of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION The present invention relates to the field of cancer. More specifically, the present invention provides compositions and methods useful for detecting long non-coding RNAs (lncRNA) in medulloblastoma. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY This application contains a sequence listing. It has been submitted electronically via EFS-Web as an ASCII text file entitled “P15814-03_ST25.txt.” The sequence listing is 55,637 bytes in size, and was created on Sep. 10, 2020. It is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION Medulloblastoma (MB), characterized as WHO group IV, represents the most common and highly malignant pediatric central nervous system tumor, representing 9.2% of all pediatric brain tumor cases and roughly 500 new cases of MB are annually diagnosed. MB are localized in the cerebellum, sharing signatures with embryonic cerebellar lineages, from where they commonly metastasize to other parts of the brain and spinal cord, and, rarely, to extraneural sites. Commonly used treatment strategies for MB, including maximal safe surgical resection, radiotherapy and chemotherapy, are aggressive for patients who are predominantly under 7 years of age. Appropriate treatment therapy selection depends upon clinical subgroup, stage, extent of resection and location, and patient's ability to withstand the treatment. To aide treatment options a combinatorial genome wide sequencing, genetic alteration and DNA methylation approach has improved MB diagnosis into four clinically and molecularly distinct subgroup: wingless (WNT) sonic hedgehog (SHH), group 3 and group 4. Despite these significant advances in early diagnosis and effective treatment approaches, MB remains a deadly disease with around 30% fatality rate. Often eradication of tumor still results in deteriorated overall quality of life due to side effects including organ dysfunction, neurocognitive impairment, endocrine disabilities, and secondary tumors. In addition, even with advances in molecular classification, the defining molecular mechanism remains unknown in group 3 and group 4, making the proper diagnosis and treatment of the respective patient challenging. Hence, there is an urgent need to identify causative molecular mechanism to drive precision medicine based approaches that could improve the quality of life of patients and increase our understanding of MB in general. SUMMARY OF THE INVENTION Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the non-coding RNA genome, in particular long non-coding RNAs (lncRNAs), contributes to MB sub-grouping. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential molecular marker and therapeutic target in group 3 MBs. Publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify lncRNAs that differentiate between MB subgroups. After characterizing a subset of differentially expressed lncRNAs in vitro and in vivo, the group 3-enriched lncRNA lnc-HLX2-7 was deleted by CRISPR/Cas9 in the MB cell line. Intracranial injected tumors were further characterized by bulk and single-cell RNA-sequencing. lnc-HLX-2-7 is highly upregulated in group 3 MB cell lines, patient-derived xenografts, and primary MBs compared to other MB sub-groups as assessed by qRT-PCR, RNA-seq, and RNA fluorescence in situ hybridization (FISH). Depletion of lnc-HLX-2-7 significantly reduced cell proliferation and 3D colony formation and induced apoptosis. lnc-HLX-2-7-deleted cells injected into mouse cerebella produced smaller tumors than those derived from parental cells. Pathway analysis revealed that lnc-HLX2-7 modulated oxidative phosphorylation, mitochondrial dysfunction, and sirtuin signaling pathways. The MY C oncogene regulated lnc-HLX-2-7 and the small molecule BET-bromodomain (BRD4) inhibitor JQ1 reduced lnc-HLX2-7 expression. lnc-HLX-2-7 is oncogenic in MB and represents a promising novel molecular marker and a potential therapeutic target in group 3 MBs. BRIEF DESCRIPTION OF THE FIGURES FIG. 1A-F. Identification and validation of the group 3-specific lncRNA, lnc-HLX-2-7. FIG. 1A: Schematic of the identification of group 3-specific lncRNAs in the four MB subgroups (WNT, SHH, group 3 and group 4). FIG. 1B: Top 50 lncRNAs with the highest expression in group 3 MBs compared to other MB subgroups are shown. x-axis indicates p-value (−log 10) of each lncRNA and y-axis indicates fold change valu