# US 11,441,195 B2 · Energetic cancer stem cells (e-CSCs): a new hyper-metabolic and proliferative tumor cell phenotype, driven by mitochondrial energy

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- Number: US 11,441,195 B2
- Document ID: US11441195B2
- Status: Granted
- Kind: B2
- Application number: 17/254,157
- Filed: 2019-06-19
- Published: 2021-08-19
- Granted: 2022-09-13
- Earliest claimed: 2018-06-19
- Assignees: Lunella Biotech, Inc.
- Applicants: Lunella Biotech, Inc.
- Inventors: Michael P. Lisanti; Federica Sotgia; Marco Fiorillo
- Web page: https://patentgrep.com/patent/US11441195B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- G01N 33/57585: {involving compounds identifiable in body fluids}
- A61K 31/03: aromatic
- A61K 31/519: ortho- or peri-condensed with heterocyclic rings
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 35/00: Antineoplastic agents
- G01N 33/57515: of the breast
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/118: Prognosis of disease development
- G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
- G01N 2800/54: Determining the risk of relapse
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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

## Abstract

This disclosure describes the characteristics of the “energetic” cancer stem cell (e-CSC) phenotype. This distinct sub-population of cancer stem cells (CSCs) has a unique energetic profile compared to bulk CSCs, being more glycolytic, having higher mitochondrial mass and elevated oxidative metabolism. e-CSCs also show an increased capacity to undergo cell cycle progression, enhanced anchorage-independent growth, and ALDH-positivity. The e-CSC phenotype presents new targets for cancer therapeutics, and in particular the anti-oxidant response, mitochondrial energy production, and mitochondrial biogenesis of e-CSCs makes them highly susceptible to mitochondrial inhibitors that target e-CSC anti-oxidant response, mitochondrial energy production, and mitochondrial biogenesis. Gene products for e-CSCs are disclosed, as well as classes of mitochondrial inhibiting therapeutic agents. Also disclosed are methods for identifying and separating e-CSCs from bulk cell populations.

## Claims

### Claim 1

A method for identifying and treating energetic cancer stem cells (e-CSCs) in a breast cancer, the method comprising:
obtaining a biological sample of the breast cancer;
determining, or having determined, a level of expression of at least one of a protein or mRNA transcript, of each member of an e-CSC gene signature comprising aldehyde dehydrogenase 5 family member A1 (ALDH5A1), thioredoxin reductase 1 (TXNR), and ribonucleotide-diphosphate reductase subunit M2 (RRM2);
comparing the determined level to a threshold level from a healthy cell line for each member of the e-CSC gene signature;
administering a pharmaceutically effective amount of at least one of an oxidative phosphorylation (OXPHOS) inhibitor and a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor if the determined level exceeds the threshold level, wherein the OXPHOS inhibitor inhibits mitochondrial function and biogenesis and the CDK4/6 inhibitor inhibits cell proliferation.

### Claim 2 (depends on 1)

The method of claim 1, wherein the gene signature comprises NAD(P)H quinone dehydrogenase 1 (NQO1), ALDH5A1, TXNR and RRM2.

### Claim 3 (depends on 1)

The method of claim 1, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises diphenyleneiodonium chloride (DPI).

### Claim 4 (depends on 1)

The method of claim 1, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises Ribociclib.

### Claim 5 (depends on 1)

The method of claim 1, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor is selected from the group consisting of DPI, atovaquone, irinotecan, sorafenib, niclosamide, berberine chloride, Ribociclib, Abemaciclib, and Palbociclib.

### Claim 6 (depends on 1)

The method of claim 1, wherein the pharmaceutically effective amount of at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor is administered if the quotient of the determined level divided by the threshold level is greater than 1.2.

### Claim 7

A method for treating tumor recurrence in a breast cancer, the method comprising:
obtaining a biological sample of the breast cancer;
performing an assay to detect the presence of e-CSCs in the biological sample using an e-CSC gene signature comprising ALDH5A1, TXNR, and RRM2, wherein the assay comprises measuring a level of expression of at least one of a protein or mRNA transcript, of each member of the e-CSC gene signature in the biological sample, and e-CSCs are detected if the measured level of expression of each member of the e-CSC gene signature exceeds a threshold level from a healthy cell line for each member of the e-CSC gene signature;
administering a pharmaceutically effective amount of at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor if e-CSCs are detected in the biological sample, wherein the OXPHOS inhibitor inhibits mitochondrial function and biogenesis and the CDK4/6 inhibitor inhibits cell proliferation.

### Claim 8 (depends on 7)

The method of claim 7, wherein the gene signature comprises NQO1, ALDH5A1, TXNR, and RRM2.

### Claim 9 (depends on 8)

The method of claim 8, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises diphenyleneiodonium chloride (DPI).

### Claim 10 (depends on 8)

The method of claim 8, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises Ribociclib.

### Claim 11 (depends on 8)

The method of claim 8, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises at least one of DPI, atovaquone, irinotecan, sorafenib, niclosamide, berberine chloride, Ribociclib, Abemaciclib, and Palbociclib.

### Claim 12 (depends on 8)

The method of claim 8, wherein the threshold level comprises a level of the at least one member from a non-cancerous biological sample.

### Claim 13 (depends on 8)

The method of claim 8, wherein the pharmaceutically effective amount of at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor is administered if the quotient of the determined level divided by the threshold level is greater than 1.2.

### Claim 14 (depends on 8)

The method of claim 8, wherein the cancer exists in a tumor that has been treated with hormone therapy.

### Claim 15

A method for treating breast cancer having one or more e-CSCs, the method comprising:
obtaining a biological sample of the breast cancer;
performing an assay to detect the presence of e-CSCs in the biological sample using an e-CSC gene signature comprising ALDH5A1, TXNR, and RRM2, wherein the assay comprises measuring a level of expression of at least one of a protein or mRNA transcript, of each member of the e-CSC gene signature in the biological sample, and e-CSCs are detected if the measured level of expression of each member of the e-CSC gene signature exceeds a threshold level from a healthy cell line for each member of the e-CSC gene signature;
administering a pharmaceutically effective amount of at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor if e-CSCs are detected in the biological sample, wherein the OXPHOS inhibitor inhibits mitochondrial function and biogenesis and the CDK4/6 inhibitor inhibits cell proliferation.

### Claim 16 (depends on 15)

The method of claim 15, wherein the gene signature comprises NQO1, ALDH5A1, TXNR and RRM2.

### Claim 17 (depends on 15)

The method of claim 15, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises diphenyleneiodonium chloride (DPI).

### Claim 18 (depends on 15)

The method of claim 15, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor comprises Ribociclib.

### Claim 19 (depends on 15)

The method of claim 15, wherein the at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor is selected from the group consisting of DPI, atovaquone, irinotecan, sorafenib, niclosamide, berberine chloride, Ribociclib, Abemaciclib, and Palbociclib.

### Claim 20 (depends on 15)

The method of claim 15, wherein the threshold level comprises a level of the at least one member from a non-cancerous biological sample.

### Claim 21 (depends on 15)

The method of claim 15, wherein the pharmaceutically effective amount of at least one of an OXPHOS inhibitor and a CDK4/6 inhibitor is administered if the quotient of the determined level divided by the threshold level is greater than 1.2.

### Claim 22 (depends on 15)

The method of claim 15, wherein the cancer exists in a tumor that has been treated with hormone therapy.

### Claim 23 (depends on 15)

The method of claim 15, wherein the breast cancer comprises at least one of a benign lesion, a pre-malignant lesion, a malignant lesion, and a metastatic lesion.

### Claim 24 (depends on 15)

The method of claim 15, further comprising administering a pharmaceutically effective amount of at least one mitochondrial inhibitor if e-CSCs are detected in the biological sample.

### Claim 25 (depends on 15)

The method of claim 15, wherein the mitochondrial inhibitor comprises at least one of a mitoriboscin, a mitoketoscin, a antimitoscin, a repurposcin, a mitoflavoscin, metformin, a tetracycline family member, a tigecycline family member, a erythromycin family member, atovaquone, bedaquiline, vitalin c, stiripentol, caffeic acid phenyl ester (CAPE), and berberine.