# US 10,767,182 B2 · Direct and selective inhibition of MDM4 for treatment of cancer

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- Number: US 10,767,182 B2
- Document ID: US10767182B2
- Status: Granted
- Kind: B2
- Application number: 15/526,716
- Filed: 2015-11-16
- Published: 2017-11-02
- Granted: 2020-09-08
- Earliest claimed: 2014-11-14
- Assignees: Vib Vzw; Katholieke Universiteit Leuven, K.U.Leuven R&D; Agency for Science, Technology and Research
- Applicants: Vib Vzw; Katholieke Universiteit Leuven, K.U.Leuven R&D; Agency for Science, Technology and Research (A*Star)
- Inventors: Jean-Christophe Marine; Ernesto Guccione; Marco Bezzi
- Web page: https://patentgrep.com/patent/US10767182B2

## CPC

- C12N 15/1135: {against oncogenes or tumor suppressor genes}
- A61K 31/428: condensed with carbocyclic rings
- A61K 31/437: the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
- A61K 31/506: not condensed and containing further heterocyclic rings
- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 35/00: Antineoplastic agents
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12N 2310/11: Antisense
- C12N 2310/3233: Morpholino-type ring
- C12N 2320/31: Combination therapy
- C12N 2320/33: Alteration of splicing
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/158: Expression markers
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present application relates to the field of cancer, particularly that of cancers with high MDM4 protein levels (such as melanoma, breast, colon or lung cancers, glioblastoma, retinoblastoma, etc.). It is shown herein that direct and selective inhibition of MDM4, e.g., by antisense RNA, leads to growth inhibition of cancer cells and sensitization to chemo or targeted therapies. Also provided are simple ways of determining which patients are most amenable for such treatment by comparing specific transcript levels.

## Claims

### Claim 1

A method of treating a subject having a tumor with an inhibitor of MDM4, the method comprising:
determining, in the tumor, or in a sample from the tumor, that the ratio of MDM4 full-length RNA transcript to MDM4 transcript lacking exon 6 is higher than 1; and
administering the inhibitor of MDM4 to the subject;
wherein the inhibitor of MDM4 induces exon 6 skipping in MDM4 mRNA.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the inhibitor acts at the RNA level.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the inhibitor is an antisense oligonucleotide.

### Claim 4 (depends on 1)

The method according to claim 1,
wherein the inhibitor of MDM4 is an antisense oligonucleotide selected from the group consisting of
an antisense oligonucleotide which binds to the exon-intron boundary of exon 6 of MDM4 and overlaps at least one Ser-Arg Splicing Factor 3 (SRSF3) binding site;
an antisense oligonucleotide which targets the 5′ donor site of the intron downstream of human MDM4 exon 6 and the adjacent downstream SRSF3 binding site;
an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 54; and
an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 60; and
wherein the antisense oligonucleotide is a morpholino or a gapmer antisense oligonucleotide.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the tumor is selected from the group consisting of stomach cancer, intestine cancer, glioblastoma, colorectal cancer, breast cancer, lung cancer, prostate cancer, osteosarcoma, retinoblastoma, melanoma, testis cancer, larynx cancer, uterus cancer, neuroblastoma, ovarian cancer, head and neck squamous cell carcinoma, and diffuse large B cell lymphoma.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the inhibitor of MDM4 is selected from the group consisting of a pharmacological or genetic inactivator of SRSF3, a short hairpin RNA (shRNA) silencer of SRSF3, meayamycin, a PRMT5 inhibitor, and a SmB inhibitor.

### Claim 7 (depends on 1)

The method according to claim 1 further comprising administering a chemotherapeutic agent or a MAPK-targeting agent to the subject.

### Claim 8

A method of treating a subject having a tumor with an inhibitor of MDM4, the method comprising:
determining, in the tumor, or in a sample from the tumor, that the ratio of MDM4 full-length RNA transcript to the sum of MDM4 transcript lacking exon 6 and MDM4 full-length RNA transcript is higher than 0.4; and
administering the inhibitor of MDM4 to the subject;
wherein the inhibitor of MDM4 induces exon 6 skipping in MDM4 mRNA.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the inhibitor acts at the RNA level.

### Claim 10 (depends on 8)

The method according to claim 8, wherein the inhibitor is an antisense oligonucleotide.

### Claim 11 (depends on 8)

The method according to claim 8,
wherein the inhibitor of MDM4 is an antisense oligonucleotide selected from the group consisting of
an antisense oligonucleotide which binds to the exon-intron boundary of exon 6 of MDM4 and overlaps at least one Ser-Arg Splicing Factor 3 (SRSF3) binding site;
an antisense oligonucleotide which targets the 5′ donor site of the intron downstream of human MDM4 exon 6 and the adjacent downstream SRSF3 binding site;
an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 54; and
an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 60; and
wherein the antisense oligonucleotide is a morpholino or a gapmer antisense oligonucleotide.

### Claim 12 (depends on 8)

The method of claim 8, wherein the inhibitor of MDM4 is selected from the group consisting of: a pharmacological or genetic inactivator of SRSF3, a short hairpin RNA (shRNA) silencer of SRSF3, meayamycin, a PRMTS inhibitor, and a SmB inhibitor.

### Claim 13 (depends on 8)

The method according to claim 8, wherein the tumor is selected from the group consisting of melanoma, stomach cancer, intestine cancer, glioblastoma, colorectal cancer, breast cancer, lung cancer, prostate cancer, osteosarcoma, retinoblastoma, testis cancer, larynx cancer, uterus cancer, neuroblastoma, ovarian cancer, head and neck squamous cell carcinoma, and diffuse large B cell lymphoma.

### Claim 14 (depends on 8)

The method according to claim 8 further comprising administering a chemotherapeutic agent or a MAPK-targeting agent to the subject.

### Claim 15

A method of treating a patient having a tumor comprising administering to the patient an antisense oligonucleotide directed to MDM4 RNA that induces skipping of exon 6 in MDM4 RNA, thereby inducing skipping of exon 6 in MDM4 RNA in a tumor of the patient.

### Claim 16 (depends on 15)

The method according to claim 15,
wherein the antisense oligonucleotide is selected from the group consisting of an antisense oligonucleotide which binds to the exon-intron boundary of exon 6 of MDM4 and overlaps at least one Ser-Arg Splicing Factor 3 (SRSF3) binding site;
an antisense oligonucleotide which targets the 5′ donor site of the intron downstream of human MDM4 exon 6 and the adjacent downstream SRSF3 binding site;
an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 54; and
an antisense oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 60; and
wherein the antisense oligonucleotide is a morpholino or a gapmer antisense oligonucleotide.

### Claim 17 (depends on 15)

The method according to claim 15, wherein the tumor is selected from the group consisting of melanoma, stomach cancer, intestine cancer, glioblastoma, colorectal cancer, breast cancer, lung cancer, prostate cancer, osteosarcoma, retinoblastoma, testis cancer, larynx cancer, uterus cancer, neuroblastoma, ovarian cancer, head and neck squamous cell carcinoma, and diffuse large B cell lymphoma.

### Claim 18 (depends on 15)

The method according to claim 15 further comprising administering a chemotherapeutic agent or a MAPK-targeting agent to the subject.