Drugs and pharmaceutical compositions

Abstract

Compounds, compositions, uses, and methods for reducing cell viability of cancer cells or for preventing or treating cancer are provided herein. In certain examples, methods for reducing cell viability of cancer cells and / or for preventing or treating cancer in a subject in need thereof are provided, which involve the use of GDP-bound Rab1a (Rab1a GDP ), Rab1a GDP The method may include the step of treatment with one or more expressible nucleic acids encoding the

Description

[Technical Field]

[0001] The present invention relates generally to the treatment of cancer. More specifically, the present invention relates to compositions and methods for enhancing microautophagy to treat various cancers and related diseases or disorders. [Background technology]

[0002] In eukaryotes, three cellular processes are known in which cytosolic contents and / or intracellular organelles can be delivered to or captured by lysosomes for degradation: macroautophagy (commonly known as autophagy), microautophagy, and chaperone-mediated autophagy. Unlike macroautophagy or chaperone-mediated autophagy, microautophagy is a type of autophagic process mediated by direct lysosomal (in mammals) or vacuolar (in plants and fungi) engulfment of cytoplasmic targets or cargo (e.g., proteins, lipids, glycogen, or pathogens) into the lysosome.

[0003] Microautophagy may contribute to cytosolic protein degradation via late endosomes (MVBs). In addition, microautophagy may also support direct glycogen delivery to lysosomes and its degradation. In this regard, it is believed that dysfunctional or insufficient microautophagy may be associated with the development of various metabolic and / or neurological disorders.

[0004] Clearly, the accumulation of specific proteins, lipids, and / or glycogen may be associated with a variety of important diseases, disorders, and conditions, and methods for targeting such proteins, lipids, and / or glycogen are desirable.

[0005] Cancer is a major ongoing health challenge, resulting in many deaths each year. It encompasses numerous types of diseases with a wide range of underlying causes. While each cancer can be biologically and pathophysiologically diverse, virtually all cancers are thought to be associated with abnormalities in membrane trafficking (Parachoniak, CA, Park, M., "Dynamics of receptor trafficking in tumorigenicity." Trends in Cell Biology, May 2012; 22(5):231-40). Unfortunately, cancer and related diseases or disorders remain a significant problem, making cancer treatments highly desirable.

[0006] Alternative, additional, and / or improved anti-cancer agents, compositions, and / or methods for treating cancer and related diseases or disorders are desirable. Summary of the Invention

[0007] As described in detail herein, GDP-bound forms of Rab1a, e.g., Rab1a S25N , Rab1a N124I , Rab1a D41N , Rab1a D47N , or another dominant-negative (DN) GDP-bound form of Rab1a, or such Rab1a GDP It has been found that treatment with microautophagy-enhancing agents comprising one or more expressible nucleic acids encoding can be used to reduce cell viability and / or cause cell death in a wide variety of different cancer cell types.

[0008] In some embodiments, there is provided herein a method for reducing cell viability of cancer cells or for preventing or treating cancer in a subject in need thereof, the method comprising: GDP-bound form of Rab1a (Rab1a GDP), Rab1a GDP treating the cancer cells of the subject with one or more expressible nucleic acids encoding This results in the production of Rab1a in cancer cells. GDP increasing intracellular levels of , resulting in decreased cell viability or death of cancer cells.

[0009] In another embodiment, there is provided herein a method for reducing cell viability of cancer cells in vitro or in vivo, the method comprising: GDP-bound form of Rab1a (Rab1a GDP ), Rab1a GDP treating the cancer cells with one or more expressible nucleic acids encoding This results in the production of Rab1a in cancer cells. GDP increasing intracellular levels of , resulting in decreased cell viability or death of cancer cells.

[0010] In another embodiment of any of the above methods, Rab1a GDP Rab1a S25N , Rab1a N124I , Rab1a D41N , Rab1a D47N , or may be or contain another dominant-negative (DN) GDP-bound form of Rab1a.

[0011] In yet another embodiment of any of the above methods, Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTD QESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; mouse Rab1 N124I ), Human Rab1a D41N or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 18; human Rab1a D47N ), Alternatively, it may include a polypeptide that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of these sequences and that preferentially binds GDP.

[0012] In yet another embodiment of any of the above methods, Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTD QESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; mouse Rab1 N124I ), Human Rab1a D41N or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 18; human Rab1a D47N ), Alternatively, it may consist of a polypeptide that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of these sequences and that preferentially binds GDP.

[0013] In another embodiment of any of the above methods, Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), Alternatively, it may comprise or consist of a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0014] In yet another embodiment of any of the above methods, Rab1a GDP may be in the form of a fusion protein, wherein Rab1a GDP is fused or linked, directly or indirectly, optionally via a linker, to a signal or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, to facilitate cellular uptake, to increase stability or in vivo half-life, or to enhance Rab1a GDP improve another therapeutic, diagnostic, or in vivo property of

[0015] In yet another embodiment of any of the above methods, the fusion protein has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), Alternatively, it may comprise a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0016] In another embodiment of any of the above methods, Rab1a GDP may be in the form of a fusion protein and have the following amino acid sequence: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDR DKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLD TGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLL IGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 21), Alternatively, it may comprise a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0017] In yet another embodiment of any of the above methods, the one or more expressible nucleic acids are Rab1a, Rab2a, Rab3a, Rab4a, Rab5a, Rab6a, Rab7a, Rab8a, Rab9a, Rab10a, Rab11a, Rab12a, Rab13a, Rab14a, Rab15a, Rab16a, Rab17a, Rab18a, Rab19a, Rab GDP It may encode one or more of the following:

[0018] In yet another embodiment of any of the above methods, the one or more expressible nucleic acids may be DNA-based or RNA-based.

[0019] In another embodiment of any of the above methods, the one or more expressible nucleic acids express Rab1a in the cell. GDP or wherein one or more expressible nucleic acids are integrated into the cellular genome and express Rab1a in the cell. GDP It may also be one that expresses

[0020] In yet another embodiment of any of the above methods, the one or more expressible nucleic acids is Rab1a GDP The vector may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the vector in the cell.

[0021] In yet another embodiment of any of the above methods, the one or more expressible nucleic acids comprises the following nucleic acid sequence: ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAA CTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAA CAGTGGCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTG CTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA (Human Rab1a S25N ORF codon sequence, SEQ ID NO: 5), ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTTGGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACATC AGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAACAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAG GAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGCCAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAAGAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCCATTTTTG GAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGCCGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGCTGCTGCTAA (Mouse Rab1a N124I ORF codon sequence, SEQ ID NO: 8), or ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACTCAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAA CTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAA CAGTGGCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTG CTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA (Human Rab1a D47N ORF codon sequence, SEQ ID NO: 17), or a Rab1a having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which binds preferentially to GDP. GDP a nucleic acid sequence encoding Alternatively, it may include nucleic acid sequences that are equivalent to any of the above sequences due to codon redundancy.

[0022] In another embodiment of any of the above methods, the one or more expressible nucleic acids have the following nucleic acid sequence: (SEQ ID NO: 19, DNA sequence of MG-008 ORF with 5' luciferase tag), or (SEQ ID NO: 20, mRNA sequence of MG-008 ORF with 5' luciferase tag), or a Rab1a having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which binds preferentially to GDP. GDP a nucleic acid sequence encoding Alternatively, it may include nucleic acid sequences that are equivalent to any of the above sequences due to codon redundancy.

[0023] In another embodiment, a method for treating or preventing cancer in a subject in need thereof includes administering to a subject a GDP-bound form of Rab1a (Rab1a GDP ), Rab1a GDP The use of one or more expressible nucleic acids encoding the nucleotides, or combinations thereof, is provided herein.

[0024] In another embodiment, provided herein is the use of the GDP-bound form of Rab1a (Rab1aGDP), one or more expressible nucleic acids encoding Rab1aGDP, or a combination thereof, to reduce cell viability of certain cancer cells in vitro or in vivo.

[0025] Another embodiment includes the use of a GDP-bound form of Rab1a (Rab1a) in the manufacture of a medicament for reducing cell viability of certain cancer cells or for preventing or treating cancer in a subject in need thereof. GDP ), Rab1a GDP The use of one or more expressible nucleic acids encoding the nucleotides, or combinations thereof, is provided herein.

[0026] Another embodiment includes the use of a GDP-bound form of Rab1a (Rab1a) in the manufacture of a medicament for reducing cell viability of certain cancer cells in vitro or in vivo. GDP), Rab1a GDP The use of one or more expressible nucleic acids encoding the nucleotides, or combinations thereof, is provided herein.

[0027] Another embodiment of any of the above uses includes Rab1a GDP Rab1a S25N , Rab1a N124I , Rab1a D41N , Rab1a D47N or another dominant negative (DN) GDP-bound form of Rab1a.

[0028] In yet another embodiment of any of the above uses, Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTD QESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; mouse Rab1 N124I ), Human Rab1a D41N or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 18; human Rab1a D47N ), Alternatively, it may include a polypeptide that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of these sequences and that preferentially binds GDP.

[0029] In yet another embodiment of any of the above uses, Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTD QESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; mouse Rab1 N124I ), Human Rab1a D41N or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 18; human Rab1a D47N ), Alternatively, it may consist of a polypeptide that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of these sequences and that preferentially binds GDP.

[0030] Another embodiment of any of the above uses includes Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), Alternatively, it may comprise or consist of a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0031] In yet another embodiment of any of the above uses, Rab1a GDP may be in the form of a fusion protein, wherein Rab1a GDP is fused or otherwise linked, directly or indirectly, optionally via a linker, to a signal or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, to facilitate cellular uptake, to increase stability or in vivo half-life, or to enhance Rab1a GDP improve another therapeutic, diagnostic, or in vivo property of

[0032] In yet another embodiment of any of the above uses, the fusion protein has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), Alternatively, it may comprise a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0033] Another embodiment of any of the above uses includes Rab1a GDP may be in the form of a fusion protein and have the following amino acid sequence: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDR DKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLD TGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLL IGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 21), Alternatively, it may comprise a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0034] In yet another embodiment of any of the above uses, the one or more expressible nucleic acids comprise one or more Rab1a as defined herein. GDP may be coded.

[0035] In yet another embodiment of any of the above uses, the one or more expressible nucleic acids may be DNA-based or RNA-based.

[0036] In another embodiment of any of the above uses, the one or more expressible nucleic acids express Rab1a in the cell. GDP or wherein one or more expressible nucleic acids are integrated into the cellular genome and express Rab1a in the cell. GDP may be expressed.

[0037] In another embodiment of any of the above uses, the one or more expressible nucleic acids is Rab1a GDP The vector may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the vector in the cell.

[0038] In another embodiment of any of the above uses, the one or more expressible nucleic acids comprises the following nucleic acid sequence: ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAA CTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAA CAGTGGCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTG CTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA (Human Rab1a S25N ORF codon sequence, SEQ ID NO: 5), ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTTGGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACATC AGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAACAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAG GAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGCCAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAAGAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCCATTTTTG GAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGCCGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGCTGCTGCTAA (Mouse Rab1a N124I ORF codon sequence, SEQ ID NO: 8), or ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACTCAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAA CTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAA CAGTGGCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTG CTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA (Human Rab1a D47N ORF codon sequence, SEQ ID NO: 17), or a Rab1a having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which binds preferentially to GDP. GDP a nucleic acid sequence encoding Alternatively, it may include nucleic acid sequences that are equivalent to any of the above sequences due to codon redundancy.

[0039] In yet another embodiment of any of the above uses, the one or more expressible nucleic acids comprise the nucleic acid sequence: (SEQ ID NO: 19, DNA sequence of MG-008 ORF with 5' luciferase tag), or (SEQ ID NO: 20, mRNA sequence of MG-008 ORF with 5' luciferase tag), or a Rab1a having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which binds preferentially to GDP. GDP a nucleic acid sequence encoding Alternatively, it may include nucleic acid sequences that are equivalent to any of the above sequences due to codon redundancy.

[0040] In another embodiment, the amino acid sequence is: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTD QESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; mouse Rab1 N124I ), Human Rab1a D41N or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 18; human Rab1a D47N ), a polypeptide comprising or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to any of these sequences and which preferentially binds GDP; Provided herein are polypeptides for reducing cell viability of specific cancer cells in a subject in need thereof, or for preventing or treating cancer, or for reducing cell viability of specific cancer cells in vitro or in vivo.

[0041] In another embodiment, provided herein is a pharmaceutical composition comprising: GDP-bound form of Rab1a (Rab1a GDP ), Rab1a GDP or a combination thereof, and Another anti-cancer drug.

[0042] In yet another embodiment, provided herein is a kit comprising any one or more of the following: GDP-bound form of Rab1a (Rab1a GDP ), Rab1a GDP one or more expressible nucleic acids encoding anticancer drugs, instructions for carrying out any of the methods described herein; or Any combination of them.

[0043] In yet another embodiment of any of the above methods, uses, or polypeptides for use, the cancer may be brain cancer, breast cancer, cervical cancer, colon cancer, ductal carcinoma, gastric cancer, liver cancer, lung cancer, oral cancer, pancreatic cancer, or prostate cancer.

[0044] In yet another embodiment of any of the above methods, uses, or polypeptides for use, the cancer may be breast adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, colorectal carcinoma, prostate cancer, embryonal rhabdomyosarcoma, gastric carcinoma, glioblastoma, hepatocellular carcinoma, invasive ductal carcinoma of the breast, lung adenocarcinoma, pancreatic ductal adenocarcinoma, papillomavirus-associated cervical adenocarcinoma, oral squamous cell carcinoma, or tongue squamous cell carcinoma.

[0045] In another embodiment of any of the above methods, uses, or polypeptides for use, the cancer cell line is selected from the group consisting of A549 (lung adenocarcinoma), HCT116 (colon carcinoma), HT29 (colon adenocarcinoma), HuCCT1 (cholangiocarcinoma), PC-3 (prostate cancer), RD (rhabdomyosarcoma), SC-M1 (gastric cancer), U-87MG (glioblastoma), SW480 (colon adenocarcinoma), PANC-1 (pancreatic adenocarcinoma), OECM-1 (oral carcinoma), OC2 (oral carcinoma), MIA (prostate cancer), and others. The cells may be selected from the group consisting of PaCa-2 (pancreatic adenocarcinoma), MDA-MB-468 (breast adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), MCF-7 (ductal carcinoma), Mahlavu (hepatocellular carcinoma), HSC-3 cells (squamous cell carcinoma of the tongue), HeLa (cervical carcinoma), HCT116 (colon carcinoma), and U-87MG (glioblastoma). [Brief explanation of the drawings]

[0046] These and other characteristics suggest that various cancer cells express the Rab1a mRNA. GDP This is further understood in connection with the following drawings, which are addressed by:

[0047] [Figure 1]Figure 1 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in A549 cells (lung adenocarcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 2] Figure 2 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in HCT116 cells (colon cancer) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 3] Figure 3 shows that transfection of 100 ng of mRNA encoding MG-008 per well induced cell death in HT29 cells (colon adenocarcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 4] Figure 4 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in HuCCT1 cells (cholangiocarcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 5]Figure 5 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in PC-3 cells (prostate cancer) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 6] Figure 6 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in RD cells (rhabdomyosarcoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 7] Figure 7 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in SC-M1 cells (gastric cancer) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 8] Figure 8 shows that transfection of Vero cells (a non-cancerous cell line) with mRNA encoding MG-008 (100 ng per well) did not result in cell death, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D) as described in Example 1, 72 hours after transfection. [Figure 9]Figure 9 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in U-87MG cells (glioblastoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 10] Figure 10 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in SW480 cells (colon adenocarcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 11] Figure 11 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in PANC-1 cells (pancreatic adenocarcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 12] Figure 12 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in OECM-1 cells (oral carcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 13]Figure 13 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in OC2 cells (oral carcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (G-008)) and crystal violet staining (D), as described in Example 1. [Figure 14] Figure 14 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in MIA PaCa-2 cells (pancreatic adenocarcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 15] Figure 15 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in MDA-MB-468 cells (breast adenocarcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 16] Figure 16 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in MDA-MB-231 cells (breast adenocarcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 17]Figure 17 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in MCF-7 cells (a breast ductal carcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 18] Figure 18 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in Mahlavu cells (hepatocellular carcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 19] Figure 19 shows that transfection of mRNA encoding MG-008 (100 ng per well) induced cell death in HSC-3 cells (squamous cell carcinoma of the tongue) 72 hours after transfection, as confirmed by phase contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 20] Figure 20 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in HeLa cells (cervical carcinoma) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 21]Figure 21 shows that transfection of MG-008-encoding mRNA (100 ng per well) induced cell death in HCT116 cells (colon cancer) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine MessengerMAX), and C (MG-008)) and crystal violet staining (D), as described in Example 1. [Figure 22] Figure 22 shows that transfection of plasmid DNA (100 ng per well) encoding MG-008 or MG-008-Luc did not cause cell death in Vero cells (a noncancerous cell line), as confirmed 48 hours after transfection by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 23] Figure 23 shows that transfection of U-87MG cells (glioblastoma) with plasmid DNA (100 ng per well) encoding MG-008 or MG-008-Luc resulted in cell death 48 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 24] Figure 24 shows that transfection of MG-008 or MG-008-Luc-encoding plasmid DNA (100 ng per well) induced cell death in SW480 cells (colon adenocarcinoma) 48 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 25]Figure 25 shows that transfection of MG-008 or MG-008-Luc-encoding plasmid DNA (100 ng per well) induced cell death in SC-M1 cells (gastric cancer) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 26] Figure 26 shows that transfection of MG-008 or MG-008-Luc-encoding plasmid DNA (100 ng per well) induced cell death in PC-3 cells (prostate cancer) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 27] Figure 27 shows that transfection of MG-008 or MG-008-Luc-encoding plasmid DNA (100 ng per well) induced cell death in PANC-1 cells (pancreatic adenocarcinoma) 48 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 28] Figure 28 shows that transfection of MG-008 or MG-008-Luc-encoding plasmid DNA (100 ng per well) induced cell death in MIA PaCa-2 cells (pancreatic adenocarcinoma) 48 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 29]Figure 29 shows that transfection of MG-008 or MG-008-Luc-encoding plasmid DNA (100 ng per well) induced cell death in MDA-MB-468 cells (breast adenocarcinoma) 48 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 30] Figure 30 shows that transfection of MG-008 or MG-008-Luc-encoding plasmid DNA (100 ng per well) induced cell death in MDA-MB-231 cells (breast adenocarcinoma) 48 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 31] Figure 31 shows that transfection of MG-008 or MG-008-Luc-encoding plasmid DNA (100 ng per well) induced cell death in MCF-7 cells (a breast ductal carcinoma) 48 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 32] Figure 32 shows that transfection of MG-008 or MG-008-Luc-encoding plasmid DNA (100 ng per well) induced cell death in Mahlavu cells (hepatocellular carcinoma) 48 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 33]Figure 33 shows that transfection of HeLa cells (cervical carcinoma) with plasmid DNA (100 ng per well) encoding MG-008 or MG-008-Luc resulted in cell death, as confirmed 48 hours after transfection by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 34] Figure 34 shows that transfection of MG-008 or MG-008-Luc-encoding plasmid DNA (100 ng per well) induced cell death in HCT116 cells (colon cancer) 72 hours after transfection, as confirmed by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 35] Figure 35 shows that transfection of A549 cells (lung adenocarcinoma) with plasmid DNA (100 ng per well) encoding MG-008 or MG-008-Luc resulted in cell death, as confirmed 72 hours after transfection by phase-contrast microscopy (panels A (control), B (Lipofectamine 2000), C (+MG-008), and D (+MG-008-Luc)) and crystal violet staining (E), as described in Example 1. [Figure 36A] FIG. 36A shows certain amino acid or nucleic acid sequences described in this application. [Figure 36B] Figure 36B shows certain amino acid or nucleic acid sequences described in this application. [Figure 36C] Figure 36C shows certain amino acid or nucleic acid sequences described in this application. [Figure 36D] Figure 36D shows certain amino acid or nucleic acid sequences described in this application. [Figure 36E]Figure 36E shows certain amino acid or nucleic acid sequences described in this application. [Figure 36F] Figure 36F shows certain amino acid or nucleic acid sequences described in this application. [Figure 36G] Figure 36G shows certain amino acid or nucleic acid sequences described in this application. [Figure 36H] Figure 36H shows specific amino acid or nucleic acid sequences described in this application. [Figure 36I] Figure 36I shows certain amino acid or nucleic acid sequences described in this application. [Figure 36J] Figure 36J shows certain amino acid or nucleic acid sequences described in this application. [Figure 36K] Figure 36K shows certain amino acid or nucleic acid sequences described in this application. [Figure 36L] Figure 36L shows certain amino acid or nucleic acid sequences described in this application. [Figure 36M] Figure 36M shows certain amino acid or nucleic acid sequences described in this application. [Figure 36N] Figure 36N shows certain amino acid or nucleic acid sequences described in this application. [Figure 36O] Figure 36O shows certain amino acid or nucleic acid sequences described in this application. [Figure 36P] Figure 36P shows certain amino acid or nucleic acid sequences described in this application. [Figure 36Q] Figure 36Q shows certain amino acid or nucleic acid sequences described in this application. [Figure 36R] Figure 36R shows certain amino acid or nucleic acid sequences described in this application. [Figure 36S] Figure 36S shows certain amino acid or nucleic acid sequences described in this application. [Figure 36T] Figure 36T shows certain amino acid or nucleic acid sequences described in this application. [Figure 36U] Figure 36U shows certain amino acid or nucleic acid sequences described in this application. [Figure 37]Figure 37 shows a female Balb / c mouse (7 weeks old) injected with 1 x 10 4T1-Fluc-Neo cells (a luciferase-expressing mouse breast cancer cell line) into the left fourth inguinal mammary gland. One week later, the mouse received a single intravenous (IV) dose of 0.05 mpk (in 100 μl PBS) of F11. Untreated control mice were injected with PBS only. In vivo imaging system (IVIS) images of bioluminescence from the 4T1 tumor cells were then recorded weekly for the next 7 consecutive weeks. [Figure 38] Figure 38 shows quantification of total luciferin flux from the experiment in Figure 37. Untreated control mice developed metastases on day 30 (Figure 38A, left panel) and died on day 37. No metastases were observed in F11-injected mice throughout the 7-week experiment (Figure 38B, right panel). [Figure 39] Figure 39 shows female Balb / c mice injected with 4T1 tumor cells into the left inguinal fourth mammary gland as described in Figure 37. One week later, the mice received a single intravenous (IV) dose of F11, and control mice were injected with PBS only. Morphological changes at the 4T1 injection site were monitored. In control mice, tumor masses were palpable 14 days after IV treatment and continued to grow until the mice died on day 35 (images in the upper panel). In F11-treated mice, tumor masses were not detected until day 35 after IV treatment (images in the lower panel). [Figure 40] Figure 40 shows the weight change of Balb / c mice after being injected with 4T1 tumor cells (as described above for Figure 37) and one week later (day 0) administered a single dose of either intravenously (IV) F11 or PBS (control). Body weights were measured until the end of the experiment (a total of 7 weeks after IV treatment). The weight of PBS control mice did not change significantly until death on day 35 (blue line). F11-treated mice gained weight throughout the experiment (orange line). DETAILED DESCRIPTION OF THE INVENTION

[0048] Described herein are compounds, compositions, uses, and methods for reducing cell viability of cancer cells in vitro or in vivo, and / or for preventing or treating cancer in a cell or subject in need thereof. It will be understood that the embodiments and examples are provided for illustrative purposes intended for those skilled in the art and are not intended to be limiting in any way.

[0049] Cancer is a major ongoing health challenge, resulting in many deaths each year. It encompasses a large group of diseases with a wide range of underlying causes. While each cancer may differ in biology and pathophysiology, virtually all cancers are thought to be associated with impaired membrane trafficking control (Parachoniak CA, Park M. "Dynamics of receptor trafficking in tumorigenicity." Trends in Cell Biology, May 2012; 22(5):231-40). In cancer initiation and progression, mitochondria and lysosomes have important roles due to their involvement in energy homeostasis and cell death (Anderson RG, Ghiraldeli LP, Pardee TS, "Mitochondria in cancer metabolism, an organelle whose time has come?" Biochimica et Biophysica Acta: Reviews on Cancer 2018 August; 1870(1):96-102). Indeed, dysregulation of lysosomal function has been found to play a key role in tumorigenesis and progression of human cancers (Davidson SM, Vander Heiden MG, "Critical Functions of the Lysosome in Cancer Biology," Annual Review of Pharmacology and Toxicology, January 6, 2017; Vol. 57: pp. 481-507).

[0050] Cancer development and progression induce fundamental changes in lysosomal function, which in turn have profound effects on tumor invasion, metastasis, and susceptibility to anticancer treatment. Rapidly dividing cells, such as cancer cells, are highly dependent on lysosomal function, and dramatic changes in lysosomal volume, composition, and subcellular localization occur during transformation and cancer progression (Zhitomirsky B, Assaraf YG, "Lysosomes as mediators of drug resistance in cancer," Drug Resistance Updates, January 2016; Vol. 24: pp. 23-33). For example, the lysosomal protease cathepsin plays an important role in cancer progression. Extracellular cathepsin activity has been linked to tumor growth and invasion, and intracellular such activity has been linked to tumor growth inhibition (Rudzinska M, Parodi A, Soond SM, Vinarov AZ, Korolev DO, Morozov AO, Daglioglu C, Tutar Y, Zamyatnin AA Jr. "The Role of Cysteine ​​Cathepsins in Cancer Progression and Drug Resistance." International Journal of Molecular Sciences 2019;20(14):3602;23 July 2019).

[0051] Autophagy is the primary intracellular degradation system, and its degradative capacity is derived from lysosomes. It is a homeostatic and evolutionarily conserved mechanism of self-digestion, by which cells degrade and recycle long-lived proteins and excess or damaged organelles. Autophagy is a highly regulated process by which misfolded proteins and organelles reach lysosomes for their degradation (Kim KH, Lee MS, "Autophagy—a key player in cellular and body metabolism," Nature Reviews Endocrinology, June 2014; 10(6):322-37). There are three different types of autophagy: macroautophagy (commonly known as autophagy), chaperone-mediated autophagy, and microautophagy. Lysosomal degradation of substrates in chaperone-mediated autophagy and microautophagy occurs directly within the lysosome (Parzych KR, Klionsky DJ, "An overview of autophagy: morphology, mechanism, and regulation." Antioxidants & Redox Signaling, 2014;20(3):460-73). The first evidence of an inhibitory role of autophagy in cancer was the observation that heterozygous loss of the gene encoding Beclin 1 (Becn1) was associated with chromosomal instability in breast, ovarian, and prostate cancers. During macroautophagy, the cytoplasm, proteins, and organelles of cancer cells are enclosed by a double-walled membrane known as an autophagosome vesicle. Subsequently, the autophagosome vesicle fuses with a lysosome to form an autophagolysosome.These contents are then degraded by lysosomal enzymes. (Gong C, Bauvy C, Tonelli G, Yue W, Delomenie C, Nicolas V, Zhu Y, Domergue V, Marin-Esteban V, Tharinger H, Delbos L, Gary-Gouy H, Morel AP, Ghavami S, Song E, Codogno P, Mehrpour M. Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like / progenitor cells. "Autophagy and the Cell Cycle: A Complex Landscape," Oncogene, May 2, 2013; 32(18): 2261-72, 2272e.1-11. Additionally, macroautophagy has been shown to activate and mediate senescence in primary biliary cirrhosis and cultured human lung fibroblasts by inducing cell cycle arrest in transformed cells, preventing tumorigenesis (Mathiassen, SG, De Zio, D, Cecconi, F, "Autophagy and the Cell Cycle: A Complex Landscape," Frontiers in Oncology, March 31, 2017; 7: 51).

[0052] In certain conditions, macroautophagy can work with mitotic catastrophe to remove cancer cells that escape apoptosis (Simon HU, Friis R, "ATG5: a distinct role in the nucleus," Autophagy 2014 January; 10(1): 176-7). As a tumor suppressor mechanism, macroautophagy maintains genomic stability and also induces senescence and potentially autophagic cell death (Gozuacik D, Kimchi A, "Autophagy as a cell death and tumor suppressor mechanism," Oncogene 2004 April 12; 23(16): 2891-906). In addition to autophagic cell death, the tumor suppressor mechanism of macroautophagy may encompass some or all of the following aspects: inhibiting abnormal cell proliferation, inhibiting chronic necrosis of cancer cells, and reducing DNA damage (Pathania AS, Guru SK, Kumar S, Kumar A, Ahmad M, Bhushan S, Sharma PR, Mahajan P, Shah BA, Sharma S, Nargotra A, Vishwakarma R, Korkaya H, Malik F, "Interplay between cell cycle and autophagy induced by boswellic acid analog," Scientific Reports Reports) September 29, 2016; Vol. 6: p. 33146).

[0053] Cell death can involve or involve different factors, such as changes in cell morphology or their function. There are three major types of cell death: apoptosis (programmed cell death type 1), autophagy (programmed cell death type 2), and necroptosis (programmed cell death type 3) (Fuchs Y, Steller H, "Programmed cell death in animal development and disease," Cell, November 11, 2011; Vol. 147(4): pp. 742-58). Generally, autophagy functions in cellular defense events. However, it can also be used as a cell suicide mechanism. This is known as "autophagic cell death." Therefore, the autophagic cell death process is distinct from apoptotic or necroptotic programmed cell death (Kroemer G, Levine B. "Autophagic cell death: the story of a misnomer." Nature Reviews Molecular Cell Biology, December 2008; 9(12):1004-10). In addition, autophagic cell death is the primary mechanism associated with the tumor-suppressive effects of macroautophagy.In this regard, myeloma cells may avoid cell death by limiting autophagy activity through cleavage of the autophagy inducer BCL2-interacting protein BCLAF1 by caspase-10 (Rosebeck S, Alonge MM, Kandarpa M, Mayampurath A, Volchenboum SL, Jasielec J, Dytfeld D, Maxwell SP, Kraftson SJ, McCauley D, Shacham S, Kauffman M, Jakubowiak AJ. Synergistic Myeloma Cell Death via Novel Intracellular Activation of Caspase-10-Dependent Apoptosis by Carfilzomib and Selinexor. "Intracellular Activation of Caspase-10-Dependent Apoptosis by Carfilzomib and Selinexor," Molecular Cancer Therapeutics, January 2016; 15(1):60-71.Autophagic cell death is frequently induced in cells lacking apoptotic mechanisms, such as p53-deficient cancer cells (Scherz-Shouval R, Weidberg H, Gonen C, Wilder S, Elazar Z, Oren M. "p53-dependent regulation of autophagy protein LC3 supports cancer cell survival under prolonged starvation." Proceedings of the National Academy of Sciences of the United States of America, 2010; 107(43):18511-6).

[0054] Reactive oxygen species (ROS) have been shown to play an important role in autophagic cell death. Excessive ROS production promotes the release of cytochrome c from mitochondria into the cytoplasm, inducing programmed cell death (Filomeni G, De Zio D, Cecconi F. "Oxidative stress and autophagy: the clash between damage and metabolic needs." Cell Death & Differentiation, March 2015; 22(3):377-88). Elevated basal levels of ROS have been observed in cancer cells, and high endogenous ROS levels in cancer cells make them vulnerable and sensitive to ROS-induced cell death (Liou GY, Storz P, "Reactive oxygen species in cancer," Free Radical Research, May 2010; Vol. 44(5): pp. 479-96). The reasons for increased ROS production in cancer cells are not fully understood, but it is generally believed to be most likely related to increased cellular bioenergetics. Because proliferating cancer cells require large amounts of energy (i.e., ATP), bioenergetic processes are massively upregulated to maintain cell proliferation, differentiation, and migration (i.e., metastasis). Unfortunately, ROS are an unavoidable by-product of bioenergetic processes, which are most likely produced in mitochondria during the ATP synthesis process known as oxidative phosphorylation.

[0055] Reactive oxygen species (ROS) generally include chemically reactive molecules containing oxygen, which can be produced as a result of cellular metabolism. Reactive oxygen species (ROS) include a group of ions and molecules, such as the hydroxyl radical (·OH), alkoxy radicals, superoxide anion (O₂·-), singlet oxygen (₁O₂), and hydrogen peroxide (H₂O₂) (Auten, RL, Davis, JM, "Oxygen toxicity and reactive oxygen species: the devil is in the details," Pediatric Research, August 2009; 66(2):121-7). Many anticancer therapies have been developed based on the ability of ROS to kill cancer cells. This includes, inter alia, for example, methoxyestradiol, buthionine sulfoximine, imexon, cisplatin, doxorubicin, motexafine gadolinium, tert-butylhydroquinone, and seleno compounds (Perillo B, Di Donato M, Pezone A, Di Zazzo E, Giovannelli P, Galasso G, Castoria G, Migliaccio A. "ROS in cancer therapy: the bright side of the moon." Experimental & Molecular Medicine 2020 February; 52(2): 192-203). Elevated ROS levels in cancer cells can result from increased ROS production or decreased ROS scavenging, or both. In both cases, endolysosomal activity is intricately involved in maintaining cellular ROS homeostasis, especially in the context of cancer.

[0056] Tumors often arise from sites of chronic irritation, infection, or inflammation. Virtually all cancer cells are thought to originate from functionally defective cells with metabolic reprogramming and elevated baseline ROS production. At the cell biological level, modulation of cellular macroautophagy processes has been observed in several cancers. Macroautophagy regulatory mechanisms also decline with age and environmental / nutritional stress, resulting in more dysfunctional or cancerous cells (Martinez-Lopez N, Athonvarangkul D, Singh R. "Autophagy and aging." Advances in Experimental Medicine and Biology 2015; 847: 73-87). Cancer cells often have significantly lower basal macroautophagy activity than normal cells. Many oncogenes and tumor suppressor genes are closely associated with macroautophagy. For example, the well-known PTEN tumor suppressor gene blocks PI3K / Akt and thus activates macroautophagy. PTEN mutations, which are very commonly found in cancer, are associated with reduced macroautophagy levels and increased cancer risk (Tan MH, Mester L, Ngeow J, Rybicki LA, Orloff MS, Eng C. "Lifetime cancer risks in individuals with germline PTEN mutations." Clinical Cancer Research 2012;15;18(2):400-7). Interestingly, many anticancer drugs have also been reported to induce macroautophagy.Some anticancer drugs that can induce macroautophagy are tamoxifen, rapamycin, arsenic trioxide, temozolomide, histone deacetylase inhibitors, ionizing radiation, vitamin D analogs, and etoposide. (Liu EY, Xu N, O'Prey J, Lao LY, Joshi S, Long JS, O'Prey M, Croft DR, Beaumatin F, Baudot AD, Mrschtik M, Rosenfeldt M, Zhang Y, Gillespie DA, Ryan KM. "Loss of autophagy causes a synthetic lethal deficiency in DNA repair.") "Repair," Proceedings of the National Academy of Sciences of the United States of America, January 20, 2015; Vol. 112(3): pp. 773-8.

[0057] In this regard, the present inventors have recognized that in addition to apoptosis (programmed cell death) and necroptosis, the process of autophagy-lysosome-mediated cell death, particularly microautophagy, can be used to develop effective strategies for anti-cancer therapy.

[0058] Stimulation of microautophagy (which in certain embodiments may involve engulfment of target membrane fragments by promoting lysosome movement to the target membrane) may be particularly desirable for the treatment of various diseases and / or disorders and / or conditions. Specific lysosomal location within a cell may be associated with different types of lysosomal activity. Additionally, lysosomal location may correlate with mTOR activity and regulate autophagic flux. During macroautophagy, mTORC1 is inactive, and lysosomes may accumulate in the perinuclear region of the cell. This may facilitate macroautophagy by stimulating fusion of the encased target membrane with lysosomes. In contrast, in the process of microautophagy, engulfment of target membrane fragments by lysosomes may occur through lysosomal movement and direct interaction with the target membrane, without the need for autophagosome formation. Lysosomes can move in a bidirectional manner on microtubules. Such lysosomal motility is governed by different sets of motor proteins recruited by different mechanisms.

[0059] In certain embodiments, microautophagy can involve the direct engulfment of cytoplasmic cargo at the limiting membrane by autophagic tubes, which can mediate both invagination into the lysosomal lumen and vesicle scission (see Li, W.-W., Li, J., and Bao, J.-K., "Microautophagy: lesser-known self-eating," Cellular and Molecular Life Sciences, 69:1125-1136 (2011)). Direct lysosomal degradation of target substrates can occur, for example, for DNA (called piecemeal autophagy; see Fujiwara, Y. et al., "Direct uptake and degradation of DNA by lysosomes" - PubMed - NCBI. Autophagy 9, 1167-1171 (2014)).In addition, lysosomes may be capable of moving to different organelles and / or membrane substrates (e.g., plasma membrane, mitochondria) through the recruitment of motor and SNARE proteins, and may interact directly with them (Andrews, NW, "Lysosomes and the plasma membrane," Journal of Cell Biology, 158, 389-394 (2002); Hofmann, I. and Munro, S., "An N-terminally acetylated Arf-like GTPase is localized to lysosomes and affects their motility," Journal of Cell Science, 1999). Science, Vol. 119, pp. 1494-1503 (2006); Fraldi, A. et al., "Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders," The EMBO Journal, Vol. 29, pp. 3607-3620 (2010); and Pankiv, S. et al., "FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport," Journal of Cell Biology. (See Biology, Vol. 188, pp. 253-269 (2010)).The movement of lysosomes to the cell edge and their positioning within the cell can be related to signal transduction (see R. and Bonifacino, J.S., “Lysosome Positioning Influences mTORC2 and AKT Signaling,” Molecular Cell 75, pp. 26-38, e3 (2019)). In this regard, it is contemplated that mTORC1, mTORC2, and AKT activation may be important for lysosome peripheral distribution (see Pous, C. and Codogno, P., “Lysosome positioning coordinates mTORC1 activity and autophagy,” Nature Cell Biology 13:342-344 (2011); and Cabukusta, B. and Neefjes, J., “Mechanisms of lysosomal positioning and movement,” Traffic 19:761-769 (2018)).

[0060] In certain embodiments, microautophagy and / or peumaric degradation of target substrates and / or membranes can involve movement of lysosomes to the periphery in the cytosol and to the target membrane (e.g., direct interaction with the target membrane) (Pu, J., Guardia, C. M., Keren-Kaplan, T., and Bonifacino, J. S., "Mechanisms and functions of lysosome positioning," Journal of Cell Science, 129, pp. 4329-4339 (2016); and Katherine R. Parzych, D. J. K., "An Overview of Autophagy: Morphology, Mechanism, and Regulation," Antioxidants & Redox Signaling, vol. 129, pp. 4329-4339 (2016)). Signaling, Vol. 20, pp. 460-473 (2014).In certain embodiments, the movement of lysosomes to the cell periphery (from the perinuclear region of the cell) and their interaction with target membranes / substrates (e.g., glycogen, lipids, proteins) at the cell periphery can be associated with the activation of mTORC1 / mTORC2 (Rabanal-Ruiz, Y. and Korolchuk, VI, "mTORC1 and Nutrient Homeostasis: The Central Role of the Lysosome," International Journal of Molecular Sciences, vol. 19, p. 818 (2018); Jia, R. and Bonifacino, JS, "Lysosome Positioning Influences mTORC2 and AKT Signaling," Molecular Cell, vol. 19, p. 818 (2018)). (See Cell) Vol. 75, p.26-38.e3 (2019)).

[0061] Without wishing to be bound by theory, it is contemplated that Rab1aDN (a dominant-negative form of Rab1a) may stimulate the peripheral distribution of lysosomes (from the perinuclear region) by activating mTORC1 / mTORC2 proteins within the cell, without the need for external (or extracellular) signals for their activation (to support the peripheral distribution of lysosomes).

[0062] Rab1a reduces cancer cell viability and / or causes cancer cell death GDP or Rab1a GTP Use of: As described in detail herein, GDP-bound forms of Rab1a, e.g., Rab1a S25N , Rab1a N124I (mouse Rab1 sequence), Rab1a D41N , Rab1a D47Nor another dominant-negative (DN) GDP-bound form of Rab1a, or such Rab1a GDP It has now been determined that treatment with a microautophagy-enhancing agent comprising one or more expressible nucleic acids encoding can be used to reduce cell viability and / or cause cell death in a wide variety of different cancer cell types.

[0063] As will be appreciated, in certain embodiments, the methods described herein may be in vitro methods, in vivo methods, or both.

[0064] In certain embodiments, provided herein is a method for reducing cell viability of cancer cells or for preventing or treating cancer in a subject in need thereof, the method comprising: GDP-bound form of Rab1a (Rab1a GDP ), Rab1a GDP treating the subject's cancer cells with one or more expressible nucleic acids encoding This results in the production of Rab1a in cancer cells. GDP Increased intracellular levels of , leading to decreased cell viability or death of cancer cells.

[0065] In another embodiment, there is provided herein a method for reducing cell viability of cancer cells in vitro or in vivo, said method comprising: GDP-bound form of Rab1a (Rab1a GDP ), Rab1a GDP treating the cancer cells with one or more expressible nucleic acids encoding This results in the production of Rab1a in cancer cells. GDP Increased intracellular levels of , leading to decreased cell viability or death of cancer cells.

[0066] In another embodiment of any of the above methods, Rab1a GDP Rab1aS25N , Rab1a N124I , Rab1a D41N , Rab1a D47N , or may be or contain another dominant-negative (DN) GDP-bound form of Rab1a.

[0067] In yet another embodiment of any of the above methods, Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTD QESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; mouse Rab1 N124I ), Human Rab1a D41N or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 18; human Rab1a D47N ), Alternatively, it may include a polypeptide that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of these sequences and that preferentially binds GDP.

[0068] In yet another embodiment of any of the above methods, Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTD QESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; mouse Rab1 N124I ), Human Rab1a D41N or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 18; human Rab1a D47N ), Alternatively, it may consist of a polypeptide that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of these sequences and that preferentially binds GDP.

[0069] In another embodiment of any of the above methods, Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), Alternatively, it may comprise or consist of a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0070] In yet another embodiment of any of the above methods, Rab1a GDP may be in the form of a fusion protein, wherein Rab1a GDP is fused or otherwise linked, directly or indirectly, optionally via a linker, to a signal or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, to facilitate cellular uptake, to increase stability or in vivo half-life, or to enhance Rab1a GDP or improve another therapeutic, diagnostic, or in vivo property of the fusion protein. In certain embodiments, which are not intended to be limiting in any way, the fusion protein may include any protein or tag for delivery to a particular or specific organ, such as GFP, YFP, mCherry, luciferase-specific antibodies, aptamers, etc.

[0071] In yet another embodiment of any of the above methods, the fusion protein has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), Alternatively, it may comprise a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0072] In another embodiment of any of the above methods, Rab1a GDP may be in the form of a fusion protein and have the following amino acid sequence: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDR DKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLD TGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLL IGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 21), Alternatively, it may comprise a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0073] In yet another embodiment of any of the above methods, the one or more expressible nucleic acids are Rab1a, Rab2a, Rab3a, Rab4a, Rab5a, Rab6a, Rab7a, Rab8a, Rab9a, Rab10a, Rab11a, Rab12a, Rab13a, Rab14a, Rab15a, Rab16a, Rab17a, Rab18a, Rab19a, Rab GDP It may encode any one or more of:

[0074] In yet another embodiment of any of the above methods, the one or more expressible nucleic acids may be DNA-based or RNA-based.

[0075] In another embodiment of any of the above methods, the one or more expressible nucleic acids express Rab1a in the cell. GDP or wherein one or more expressible nucleic acids are integrated into the cellular genome and express Rab1a in the cell. GDP can be expressed.

[0076] In yet another embodiment of any of the above methods, the one or more expressible nucleic acids is Rab1a GDP The vector may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the vector in the cell.

[0077] In yet another embodiment of any of the above methods, the one or more expressible nucleic acids comprises the following nucleic acid sequence: ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAA CTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAA CAGTGGCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTG CTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA (Human Rab1a S25N ORF codon sequence, SEQ ID NO: 5), ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTTGGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACATC AGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAACAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAG GAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGCCAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAAGAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCCATTTTTG GAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGCCGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGCTGCTGCTAA (Mouse Rab1a N124I ORF codon sequence, SEQ ID NO: 8), or ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACTCAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAA CTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAA CAGTGGCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTG CTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA (Human Rab1a D47N ORF codon sequence, SEQ ID NO: 17), or a Rab1a having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which binds preferentially to GDP. GDP a nucleic acid sequence encoding Alternatively, it may include nucleic acid sequences that are equivalent to any of the above sequences due to codon redundancy.

[0078] In another embodiment of any of the above methods, the one or more expressible nucleic acids have the following nucleic acid sequence: (SEQ ID NO: 19, DNA sequence of MG-008 ORF with 5' luciferase tag), or (SEQ ID NO: 20, mRNA sequence of MG-008 ORF with 5' luciferase tag), or a Rab1a having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which binds preferentially to GDP. GDP a nucleic acid sequence encoding Alternatively, it may include nucleic acid sequences that are equivalent to any of the above sequences due to codon redundancy.

[0079] In another embodiment, a method for treating or preventing cancer in a subject in need thereof includes administering to a subject a GDP-bound form of Rab1a (Rab1a GDP ), Rab1a GDP The use of one or more expressible nucleic acids encoding the nucleotides, or combinations thereof, is provided herein.

[0080] In another embodiment, provided herein is the use of the GDP-bound form of Rab1a (Rab1aGDP), one or more expressible nucleic acids encoding Rab1aGDP, or a combination thereof, to reduce cell viability of cancer cells in vitro or in vivo.

[0081] In another embodiment, the present invention relates to the use of a GDP-bound form of Rab1a (Rab1a) in the manufacture of a medicament for reducing cell viability of cancer cells or for preventing or treating cancer in a subject in need thereof. GDP ), Rab1a GDP The use of one or more expressible nucleic acids encoding the nucleotides, or combinations thereof, is provided herein.

[0082] Another embodiment involves the use of a GDP-bound form of Rab1a (Rab1a) in the manufacture of a medicament for reducing cell viability of cancer cells in vitro or in vivo. GDP ), Rab1a GDPThe use of one or more expressible nucleic acids encoding the nucleotides, or combinations thereof, is provided herein.

[0083] Another embodiment of any of the above uses includes Rab1a GDP Rab1a S25N , Rab1a N124I , Rab1a D41N , Rab1a D47N , or may be or contain another dominant-negative (DN) GDP-bound form of Rab1a.

[0084] In yet another embodiment of any of the above uses, Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTD QESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; mouse Rab1 N124I ), Human Rab1a D41N or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 18; human Rab1a D47N ), Alternatively, it may include a polypeptide that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of these sequences and that preferentially binds GDP.

[0085] In yet another embodiment of any of the above uses, Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTD QESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; mouse Rab1 N124I ), Human Rab1a D41N or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 18; human Rab1a D47N ), Alternatively, it may consist of a polypeptide that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of these sequences and that preferentially binds GDP.

[0086] Another embodiment of any of the above uses includes Rab1a GDP has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), Alternatively, it may comprise or consist of a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0087] In yet another embodiment of any of the above uses, Rab1a GDP may be in the form of a fusion protein, wherein Rab1a GDP is fused or otherwise linked, directly or indirectly, optionally via a linker, to a signal or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, to facilitate cellular uptake, to increase stability or in vivo half-life, or to enhance Rab1a GDP improve another therapeutic, diagnostic, or in vivo property of

[0088] In yet another embodiment of any of the above uses, the fusion protein has the following amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), Alternatively, it may comprise a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0089] Another embodiment of any of the above uses includes Rab1a GDP may be in the form of a fusion protein and have the following amino acid sequence: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDR DKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLD TGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLL IGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 21), Alternatively, it may comprise a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which preferentially binds GDP.

[0090] In yet another embodiment of any of the above uses, the one or more expressible nucleic acids comprise one or more Rab1a as defined herein. GDP may be coded.

[0091] In yet another embodiment of any of the above uses, the one or more expressible nucleic acids may be DNA-based or RNA-based.

[0092] In another embodiment of any of the above uses, the one or more expressible nucleic acids express Rab1a in the cell. GDP or wherein one or more expressible nucleic acids are integrated into the cellular genome and express Rab1a in the cell. GDP can be expressed.

[0093] In another embodiment of any of the above uses, the one or more expressible nucleic acids is Rab1a GDP The vector may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the vector in the cell.

[0094] In another embodiment of any of the above uses, the one or more expressible nucleic acids comprises the following nucleic acid sequence: ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAA CTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAA CAGTGGCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTG CTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA (Human Rab1a S25N ORF codon sequence, SEQ ID NO: 5), ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTTGGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACATC AGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAACAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAG GAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGCCAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAAGAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCCATTTTTG GAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGCCGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGCTGCTGCTAA (Mouse Rab1a N124I ORF codon sequence, SEQ ID NO: 8), or ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACTCAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAA CTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAA CAGTGGCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTG CTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA (Human Rab1a D47N ORF codon sequence, SEQ ID NO: 17), or a Rab1a having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which binds preferentially to GDP. GDP a nucleic acid sequence encoding Alternatively, it may include nucleic acid sequences that are equivalent to any of the above sequences due to codon redundancy.

[0095] In yet another embodiment of any of the above uses, the one or more expressible nucleic acids comprise the nucleic acid sequence: (SEQ ID NO: 19, DNA sequence of MG-008 ORF with 5' luciferase tag), or (SEQ ID NO: 20, mRNA sequence of MG-008 ORF with 5' luciferase tag), or a Rab1a having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto and which binds preferentially to GDP. GDP a nucleic acid sequence encoding Alternatively, it may include nucleic acid sequences that are equivalent to any of the above sequences due to codon redundancy.

[0096] In another embodiment, the amino acid sequence is: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; human Rab1a S25N ), MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTD QESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; mouse Rab1 N124I ), Human Rab1a D41N or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNV KQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 18; human Rab1a D47N ), a polypeptide comprising or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to any of these sequences and which preferentially binds GDP; Provided herein is a polypeptide for use in reducing cell viability of cancer cells or in preventing or treating cancer in a subject in need thereof, or for use in reducing cell viability of cancer cells in vitro or in vivo.

[0097] In another embodiment, provided herein is a pharmaceutical composition comprising: GDP-bound form of Rab1a (Rab1a GDP ), Rab1a GDP or a combination thereof, and Another anti-cancer drug.

[0098] In yet another embodiment, provided herein is a kit comprising any one or more of the following: GDP-bound form of Rab1a (Rab1a GDP ), Rab1a GDP one or more expressible nucleic acids encoding anticancer drugs, instructions for carrying out any of the methods described herein; or Any combination of them.

[0099] As will be understood, in certain embodiments of any of the methods, uses, or polypeptides for use described herein, the cancer or cancer cells may be any of a wide variety of cancer types. As described in Example 1 below, test results indicate that anti-cancer effects can be observed in a wide variety of different cancer cell lines, supporting broad anti-cancer applicability. Anti-cancer effects were also observed in vivo, as described in Example 2 below, further supporting applicability to the treatment of cancer in subjects.

[0100] In yet another embodiment of any of the above methods, uses, or polypeptides for use, the cancer may be brain cancer, breast cancer, cervical cancer, colon cancer, ductal carcinoma, gastric cancer, liver cancer, lung cancer, oral cancer, pancreatic cancer, or prostate cancer.

[0101] In yet another embodiment of any of the above methods, uses, or polypeptides for use, the cancer may be breast adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, colorectal carcinoma, prostate cancer, embryonal rhabdomyosarcoma, gastric carcinoma, glioblastoma, hepatocellular carcinoma, invasive ductal carcinoma of the breast, lung adenocarcinoma, pancreatic ductal adenocarcinoma, papillomavirus-associated cervical adenocarcinoma, oral squamous cell carcinoma, or tongue squamous cell carcinoma.

[0102] In another embodiment of any of the above methods, uses, or polypeptides for use, the cancer cell line may be selected from the group consisting of A549 (lung adenocarcinoma), HCT116 (colon carcinoma), HT29 (colon adenocarcinoma), HuCCT1 (cholangiocarcinoma), PC-3 (prostate cancer), RD (rhabdomyosarcoma), SC-M1 (gastric cancer), U-87MG (glioblastoma), SW480 (colon adenocarcinoma), PANC-1 (pancreatic adenocarcinoma), OECM-1 (oral carcinoma), OC2 (oral carcinoma), MIA PaCa-2 (pancreatic adenocarcinoma), MDA-MB-468 (breast adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), MCF-7 (ductal carcinoma), Mahlavu (hepatocellular carcinoma), HSC-3 (squamous cell carcinoma of the tongue), HeLa (cervical carcinoma), and HCT116 (colon carcinoma).

[0103] As will be understood, lysosome-mediated microautophagy can refer to a cellular process in which cellular lipid or protein or glycogen substrates, or portions thereof, are engulfed and degraded by lysosomes. Specifically in this regard, lysosome-mediated microautophagy is an essential component of cellular bioenergetic (i.e., ATP generation) and biosynthetic (i.e., synthesizing new biomass by recycling existing "old" material) processes.

[0104] It will also be understood that increasing lysosomal-mediated microautophagy can refer to an increase in the rate, extent, capacity, or effectiveness of the lysosomal-mediated microautophagy process in a cell compared to the baseline level of the cell, or compared to corresponding treated or untreated control cells, or compared to levels in reference diseased cells or reference cells having accumulation of protein, lipid, or glycogen substrates.

[0105] Furthermore, it will be understood that increasing lysosome-mediated microautophagy can include restoring or increasing intracellular lysosomal motility or bidirectional lysosomal motility, and / or enhancing, increasing, activating, or otherwise restoring or rescuing cellular lysosomal motility activity. Such restoration or rescuing can result in increased autophagy (e.g., micro- and / or macroautophagy) and / or lysosomal degradation capacity. As detailed herein, there are several diseases, conditions, and cellular states in which cellular lysosomal motility can be impaired, reduced, blocked, or inhibited. Because lysosome-mediated microautophagy can involve lysosomal motility and kiss-and-run events, microautophagy-enhancing agents can restore or increase intracellular lysosomal motility. Lysosome motility can play an important role in several cellular functions, including lysosome-mediated microautophagy, lysosome-mediated macroautophagy, lysosome regeneration, and lysosome maturation process.Restoring lysosome motility and / or lysosome bidirectional motility can refer to adjusting the lysosome motility / bidirectional motility level of cells to return to that of corresponding normal cells or healthy control cells with baseline level of lysosome motility / bidirectional motility.In certain embodiments, this adjustment can also adjust the degradation ability (i.e., phagy) level to return to that of normal or healthy cells.

[0106] It will be understood that a lysosome binding-dissociation event between a lysosome and a lipid, protein, or glycogen substrate can refer to an event in which the lysosome binds to a lipid, protein, or glycogen substrate (i.e., for example, a lipid droplet or protein aggregate), captures at least a portion of the lipid, protein, or glycogen substrate, and then dissociates from the lipid, protein, or glycogen substrate. A lysosome binding-dissociation (i.e., "on" and "off") event between a lysosome and a lipid, protein, or glycogen substrate can be considered a "kiss-and-run" type event. As part of the binding (or "kissing") event, at least a small piece of the substrate (i.e., lipid) can be "caught" or engulfed by the lysosome from the substrate (i.e., for example, a cytosolic lipid droplet or CLD). In the case of a lipid droplet substrate, this can be achieved by the formation of a fusion pore between the lysosome and the CLD. As part of the dissociation (or "run") event, dissociation of the lysosome from the substrate (i.e., the CLD) can occur. An increase in lysosome binding-dissociation events can refer to an increase in the rate, extent, or effectiveness of lysosome binding-dissociation events in a cell compared to the baseline levels of corresponding treated or untreated control cells, e.g., identical cells under identical conditions but without a microautophagy-modulating agent or treated with a compound or composition known not to affect the process.

[0107] It will be appreciated that in certain embodiments, microautophagy-enhancing agents may be used to correct a cellular microautophagy deficiency or a cellular condition in which microautophagy is reduced.

[0108] Those skilled in the art will understand, in conjunction with the teachings of the present application, that a microautophagy-enhancing agent can be any suitable agent that increases or promotes the rate, activity, extent, or effectiveness of lysosome-mediated microautophagy in a cell, or that increases lysosomal motility or bidirectional motility. In certain embodiments, a suitable microautophagy-enhancing agent is a GDP-bound form of Rab1a (Rab1a GDP ), Rab1a GDP The nucleic acid may be or comprise one or more expressible nucleic acids encoding the

[0109] The Ras-related protein Rab-1A (i.e., Rab1a) is a protein encoded by the RAB1A gene in humans. It can control vesicle trafficking from the endoplasmic reticulum (ER) to the Golgi compartment and then to the cell surface, and may play an important role in IL-8 and growth hormone secretion. In addition, when it is in its GTP-bound form, it may play a role in the assembly of autophagosomes in macroautophagy and in the cellular defense response against pathogenic bacteria. It can also control the motility of endocytic compartments.

[0110] As described in detail herein, lysosome-mediated microautophagy of target protein, lipid, or glycogen substrates in cells involves the lysis of GDP-bound forms of Rab1a, e.g., Rab1a S25N , Rab1a N124I , Rab1a D41N , Rab1a D47N It has now been identified that microautophagy can be increased by treatment with microautophagy enhancers, including Rab1a, or another dominant-negative (DN) GDP-bound form of Rab1a.

[0111] It will be understood that the specific amino acid or nucleic acid sequence of a particular gene may vary between species. For example, a human Rab1a amino acid sequence may have a homolog in another species that has a sequence variation from the human sequence. In some embodiments, although homologous sequences may vary between species, the general effect (e.g., phenotypic effect) of the homologous sequence may be substantially similar to the effect of the wild-type sequence in a given cell or subject.

[0112] In a specific embodiment, the microautophagy enhancer is a GDP-bound form of Rab1a (Rab1a GDP ), e.g., Rab1a S25N , Rab1a N124I , Rab1a D41N , Rab1a D47N , or a functional equivalent thereof, or another dominant negative (DN) GDP-bound form of Rab1a. The sequences of human and / or mouse Rab1aWT and their specific GDP-bound forms are shown in Figure 36. Suitable GDP-bound forms of Rab1a can include any suitable Rab1a variant that is "dominant negative" or that preferentially binds GDP over GTP. Such Rab1a GDPVariants can be identified using techniques known in the art (e.g., Chan, C.-C. et al., "Systematic Discovery of Rab GTPases with Synaptic Function in Drosophila," Current Biology, Vol. 21, pp. 1704-1715 (2011); Tabancay, A.P. et al., "Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K," Journal of Biological Chemistry, 2009; Chemistry, Vol. 278, pp. 39921-39930 (2003); and Dumas, JJ, Zhu, Z., Connolly, JL, and Lambright, DG, "Structural basis of activation and GTP hydrolysis in Rab proteins," Structure, Vol. 7, pp. 413-s2 (1999), each of which is incorporated herein by reference in its entirety.

[0113] Figures 36A-36U show specific sequences of nucleic acids and amino acids / proteins described herein. In Figures 36A-36U, SEQ ID NOS: 1-3 correspond to human Rab1a, respectively. WT The DNA gene sequence, ORF codon sequence, and amino acid sequence are provided. SEQ ID NOs: 4 to 6 are human Rab1a S25N The DNA gene sequence, ORF codon sequence, and amino acid sequence are provided. SEQ ID NOS: 7 to 9 are the sequences of mouse Rab1, N124IThe DNA gene sequence, ORF codon sequence, and amino acid sequence are provided. SEQ ID NOs: 10 to 12 are human Rab1a Q70L The DNA gene sequence, ORF codon sequence, and amino acid sequence are provided. SEQ ID NOs: 13 to 15 are human Rab1a Q63L The DNA gene sequence, ORF codon sequence, and amino acid sequence are provided. SEQ ID NOs: 16 to 18 are human Rab1a D47N The DNA gene sequence, ORF codon sequence, and amino acid sequence are provided. SEQ ID NOs: 19-21 provide the DNA sequence of the MG-008 ORF with a 5' luciferase tag, the mRNA sequence of the MG-008 ORF with a 5' luciferase tag, and the MG-008 protein sequence with an N-terminal luciferase tag, respectively (see Example 1 for further discussion of SEQ ID NOs: 19-21). Human Rab1a D41N The array of https: / / www.addgene.org / 49581 / (which is incorporated herein by reference in its entirety) and is commercially available. In certain embodiments, nucleic acids or amino acids comprising any of these sequences are provided herein. In certain embodiments, nucleic acids or amino acids having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of these sequences (i.e., any of SEQ ID NOS: 1-21 or Rab1a). D41N Provided herein are nucleic acids or amino acids comprising a nucleic acid or amino acid sequence having a sequence similar to that of the sequence of the amino acid sequence of the present invention (relative to the sequence of the amino acid sequence of the present invention), or active fragments thereof.

[0114] Rab1a S25N , Rab1a D41N , Rab1a D47N , and Rab1a N124I Suitable functional equivalents of Rab1a, for example, have at least 80% (or >85%, or >90%, or >95%, or >99%) sequence identity with Rab1a. WT or Rab1a S25N or Rab1a N124I or Rab1a D41N or Rab1aD47N and preferentially binds GDP over GTP, while interacting with Rab1a as described in detail herein. S25N or Rab1a N124I or Rab1a D41N or Rab1a D47N In a further embodiment, the microautophagy enhancer may comprise a suitable Rab1a variant or mutant that also retains the relevant cellular / biochemical function of Rab1a. GDP One or more expressible nucleic acids encoding Rab1a, e.g., the GDP-bound form of Rab1a (Rab1a GDP ), e.g., Rab1a S25N or Rab1a N124I or Rab1a D41N or Rab1a D47N It will be understood that the nucleic acid sequence may be or comprise any suitable nucleic acid / expression vector (i.e., e.g., vector, cassette, mRNA, modified mRNA, plasmid), etc. that encodes / expresses the nucleic acid sequence of the present invention, or a functional equivalent thereof.

[0115] As will be understood, sequences are described herein primarily with reference to human and / or mouse homologs. It will be understood that functional equivalents and / or variants may be found in a variety of different species, for example, in different mammals. GDP (DN) or Rab1a GTP References to modifications and / or variants of specific sequences that allow for (DA) morphology are often provided for convenience with reference to human and / or mouse homologues / sequences, providing the location and modification / mutation information (e.g., S25N, D41N, D47N, N124I; Q70L, Q67L, Q63L). However, it will be understood that equivalent DN and / or DA morphologies can be implemented in homologous sequences from other species and / or other sequences related to the human and / or mouse sequences provided herein, although the location and / or nature of the modification / mutation may vary somewhat depending on the specific sequence of interest. For example, Rab1 N124Iis a modification / mutation of the mouse sequence. Another example is Rab1 Q67L is a modification / mutation in the mouse sequence; human Rab1a does not have a Q at position 67. Rather, the human sequence has a Q at position 63, so the modification / mutation with reference to the human sequence is Rab1a. Q63L is.

[0116] Those skilled in the art will appreciate that microautophagy reducers may bind to the GTP-bound form of Rab1a (Rab1a GTP ), Rab1a GTP one or more expressible nucleic acids encoding Rab1a wild type (Rab1a WT ), or Rab1a WT It will be understood that the nucleic acid sequence may be or may comprise any one or more of one or more expressible nucleic acids encoding Rab1a. GTP Rab1a Q70L , Rab1a Q67L (mouse sequence), Rab1a Q63L (human Rab1a sequence), or a functional equivalent thereof, or another dominantly active (DA) GTP-bound form of Rab1a. The GTP-bound form of Rab1a can include any Rab1a variant that is "dominantly active" or that preferentially binds GTP over GDP. Such Rab1a GTPVariants can be identified by techniques known in the art (e.g., Chan, C.-C. et al., "Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila," Current Biology, Vol. 21, pp. 1704-1715 (2011); Tabancay, A.P. et al., "Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K," Journal of Biological Chemistry, 1999). Chemistry, Vol. 278, pp. 39921-39930 (2003); and Dumas, JJ, Zhu, Z., Connolly, JL, and Lambright, DG, "Structural basis of activation and GTP hydrolysis in Rab proteins," Structure, Vol. 7, pp. 413-s2 (1999), each of which is incorporated herein by reference in its entirety), e.g., Rab1a Q70L and / or Rab1a Q67L and / or Rab1a Q63L can be identified using the same used to identify

[0117] In certain embodiments, Rab1a GDP Treatment with microautophagy enhancers such as Rab1a GDPIntroducing proteins into cells, Rab1a GDP It will be understood that the expression of a gene may include expressing a gene that is a nucleotide sequence, a nucleotide sequence, or both.

[0118] It will be understood that expression of a specific protein in a cell can refer to the production of a polypeptide from a nucleic acid sequence encoding the polypeptide. Gene expression can encompass both transcription and translation processes, and therefore gene expression can refer to the production of a nucleic acid sequence such as mRNA (i.e., transcription), the production of a protein (i.e., translation), or both. Furthermore, it will be understood that overexpression of a specific gene in a cell can refer to increasing the expression of the specific gene in the cell compared to wild-type, baseline, or untreated levels. Introduction or overexpression of a mutant gene into a cell can be achieved using any of several methods known in the art. For example, vectors (whether viral, plasmid, or other) containing one or more copies of a specific gene, each driven by a suitable promoter sequence (e.g., a constitutive or inducible promoter), or mRNA or a chemically modified version thereof, can be introduced into a cell by transfection, electroporation, viral infection, or another suitable method known in the art. Suitable expression vector technologies for introducing or overexpressing specific genes into cells are known in the art (see, e.g., Molecular Cloning: A Laboratory Manual (4th ed.), 2012, Cold Spring Harbor Laboratory Press). In conjunction with the teachings of the present application, those skilled in the art will be able to identify Rab1a vectors that can be prepared for introduction into cells to provide expression of a protein of interest (e.g., either transiently or long-term via integration into the genome). GDP One will know a wide variety of expressible nucleic acids that encode specific proteins such as:

[0119] It will be understood that compounds and / or compositions comprising or consisting of one or more of the nucleic acids and / or proteins described herein may be used. The compositions may additionally comprise one or more pharmaceutically acceptable diluents, carriers, excipients, or buffers. The compositions may be used to administer one or more nucleic acids and / or proteins to cells in vitro or in vivo.

[0120] In the context of inserting a nucleic acid sequence into a cell, the introduction of a gene may refer to "transfection," "transformation," or "transduction," and may encompass the incorporation or introduction of a nucleic acid sequence into a eukaryotic cell, where the nucleic acid sequence may optionally be integrated into the cell's genome or transiently expressed (e.g., transfected mRNA). A protein or enzyme may be introduced into a cell by delivering the protein or enzyme itself into the cell, or by expressing an mRNA encoding the protein or enzyme within the cell, resulting in its translation.

[0121] As would be known to one of skill in the art, expressible nucleic acids for expressing a particular gene may encode or include features described in "Genes VII," Lewin, B., Oxford University Press (2000) or "Molecular Cloning: A Laboratory Manual," Sambrook et al., Cold Spring Harbor Laboratory, 3rd ed. (2001). The nucleotide sequence encoding the polypeptide or protein may be incorporated into a suitable vector, such as a commercially available vector. Vectors may also be individually constructed or modified using standard molecular biology techniques, for example, as outlined in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd ed. (2001)). One of skill in the art will recognize that a vector may include a nucleotide sequence encoding desired elements that may be operably linked to a nucleotide sequence encoding a polypeptide or protein. Such nucleotide sequences encoding desired elements may include a transcription promoter, a transcription enhancer, a transcription terminator, a translation initiation factor, a translation terminator, a ribosome binding site, a 5' untranslated region, a 3' untranslated region, a cap structure, a polyA tail, and / or an origin of replication. Selection of an appropriate vector may depend on several factors, including, without limitation, the size of the nucleic acid to be incorporated into the vector, the type of transcriptional and translational control elements desired, the desired expression level, the desired copy number, whether chromosomal integration is desired, the type of selection process desired, or the host cell or host range intended to be transformed.

[0122] Those skilled in the art will understand that the biomolecules and / or compounds described herein can be provided as pharmaceutical compositions, together with a pharmaceutically acceptable diluent, carrier, or excipient, and / or together with one or more separate active agents or drugs, as part of a combination or pharmaceutical composition. In certain embodiments, the biomolecules, compounds, and / or pharmaceutical compositions can be administered simultaneously, sequentially, or in combination with other drugs or pharmaceutical compositions in a treatment regimen, either separately or as a combination or pharmaceutical composition.

[0123] The biomolecules, compounds, and / or compositions described herein may include one or more pharmaceutically acceptable excipients, diluents, and / or carriers. Pharmaceutically acceptable carriers, diluents, or excipients may include any suitable carrier, diluent, or excipient known to those of skill in the art. Examples of pharmaceutically acceptable excipients may include, but are not limited to, cellulose derivatives, sucrose, and starch. Those skilled in the art will recognize that pharmaceutically acceptable excipients may include suitable fillers, binders, lubricants, buffers, flow agents, and disentanglers known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2006)). Examples of pharmaceutically acceptable carriers, diluents, and excipients can be found, for example, in Remington's Pharmaceutical Sciences (2000 - 20th Edition) and the United States Pharmacopeia: National Formulary (USP24 NF19), 1999.

[0124] It will also be understood that one or more conservative amino acid substitutions may be possible. As will be recognized, conservative amino acid substitutions may include replacing one amino acid with another amino acid having similar properties such that protein folding, activity, or other functionality is not significantly affected. Examples of aromatic amino acids that may be substituted include phenylalanine, tryptophan, and tyrosine. Examples of interchangeable hydrophobic amino acids that may be substituted include leucine, isoleucine, methionine, and valine. Examples of interchangeable polar amino acids that may be substituted include glutamine and asparagine. Examples of interchangeable basic amino acids that may be substituted include arginine, lysine, and histidine. Examples of interchangeable acidic amino acids that may be substituted include aspartic acid and glutamic acid. Finally, examples of interchangeable small amino acids that may be substituted include alanine, serine, threonine, cysteine, and glycine.

[0125] As described in detail herein, dominant-negative (DN) Rab1a (e.g., GDP-bound Rab1a, Rab1a GDP ) can be used to increase microautophagy, which can allow for the degradation of cellular targets of interest, such as disease-related proteins, glycogen, or lipids. As will be appreciated, any suitable Rab1aDN or Rab1a GDP may be used. In light of the teachings of the present application, a variety of Rab1a DN proteins will be known to those skilled in the art. In general, in certain embodiments, any suitable dominant-negative form of Rab1a (i.e., Rab1a fixed / locked in its GDP form) may be used to, for example, promote lysosome movement to the cytosol and periphery of the cell (from the perinuclear region of the cell) and stimulate direct lysosomal interaction with target substrates.

[0126] In certain embodiments, it is contemplated that expression of Rab1aDN (GDP form) may stimulate activation of mTORC1 / mTORC2 and AKT and promote lysosome movement to the periphery and target substrates based on their effects on lysosomal positioning (see also Jia, R. and Bonifacino, JS, "Lysosome Positioning Influences mTORC2 and AKT Signaling," Molecular Cell 75:26-38.e3 (2019)).

[0127] It is contemplated herein that different genetic modifications of Rab1a that lock this protein in its constant GDP-bound state can be used to promote direct lysosomal interaction and endocytosis of different target substrates (e.g., one or more protein, lipid, and / or glycogen targets). The constant GDP-bound form (DN) of Rab1a is not generally available under normal physiological conditions, in which the native protein Rab1a constantly shifts between its GTP and GDP forms. In certain embodiments, it is contemplated that genetic mutations and / or amino acid substitutions / modifications can be used to lock this GTPase in its GDP-bound form (or constant GTP form) and prevent it from going to its GTP state (or GDP state). In certain embodiments, it is contemplated that any suitable modification that can substantially preserve the integrity of the GDP form / state of the GTPase (which can exert effects on signal transduction and on lysosomal movement to the periphery / cytosol) can promote microautophagy and degradation of target substrates by engulfment of lysosomal piecemeal.

[0128] In certain embodiments, Rab1a GDPcan generally be administered to a particular cell type or subject in need thereof in any suitable manner, which can be selected to suit the particular cell type, subject, and / or indication. In certain embodiments, Rab1a GDP Nucleic acid sequences encoding and capable of expressing a protein can be administered to a subject or introduced into a cell type by any suitable transfection or nucleic acid delivery approach that would be known to one of skill in the art in light of the teachings of the present application. In certain embodiments, delivery can be based on DNA or RNA transfection (e.g., using common transfection reagents such as lipofectamine (Invitrogen), FuGENE (Roche)), using DNA adenovirus (gene therapy), or using modified RNA (i.e., stabilized RNA) delivery to the body (e.g., using viruses, or microvesicles, exosomes, or ectosomes). In certain embodiments, the protein can be administered or delivered to cells in need thereof, optionally assisted by any suitable technique or delivery vehicle for facilitating protein delivery to cells.

[0129] In certain embodiments, Rab1a GDP or Rab1a DN can be administered to specific cell types, such as cancer cells, using lipid nanoparticles (LNPs). LNPs target Rab1a GDP or Rab1a DN , Rab1a GDP or Rab1a DN It is anticipated that the vector will encapsulate a nucleic acid capable of expressing a nucleic acid sequence of the invention, a nucleic acid sequence capable of expressing a nucleic acid sequence of the invention, or a combination thereof.

[0130] LNP is an advanced non-viral gene delivery system.LNP allows those skilled in the art to safely and effectively deliver nucleic acid to cells in vitro or in vivo.It has applications in, but not limited to, gene editing, rapid vaccine development, immuno-oncology, and the treatment of rare genetic diseases and undruggable diseases.

[0131] LNPs are being used more frequently in the art due to their advantageous properties, including controlled and sustained release characteristics, low toxicity, biocompatibility with tissues and cells, low immune response, increased deliverable gene size, cell-free manufacturing, high nucleic acid encapsulation efficiency, potent transfection, and improved penetration into tissues where therapeutic agents are to be delivered.

[0132] LNP formulations may be any number of lipid / molecule combinations, including but not limited to, ionizable cationic lipids, neutral lipids, helper lipids, phospholipids, poly(lactic-co-glycolic acid) (PLGA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and polyethylene glycol (PEG)-lipid conjugates. The LNPs used may be custom made or purchased from commercial sources, such as, but not limited to, TriLink Biotechnologies or Precision Nanosystems.

[0133] It is anticipated that LNPs may be replaced by liposomes or polymeric nanoparticles. [Example]

[0134] Example 1: Rab1a DN (GDP form) induces cancer cell death and stimulates microautophagy A series of experiments were performed to compare Rab1a activity against a wide variety of different cancer cell types. DN We investigated the anti-cancer effects of Rab1a. DN (in this example, Rab1a S25N Rab1a DN was introduced into cells either as transfected mRNA nucleic acid or as transfected DNA expression plasmid. The latter was shown to inhibit Rab1a expression in cancer cells. S25N , or Rab1a S25N A fusion protein containing Rab1a and an N-terminal luciferase tag can be expressed.DN The Rab1a used in these studies is also known as MG-008. S25N The sequence of the ORF used in the DNA expression plasmid expressing the fusion protein containing the Rab1a and N-terminal luciferase tags is shown in Figure 36S as SEQ ID NO: 19. S25N and the sequence of the ORF of the mRNA expressing the fusion protein containing an N-terminal luciferase tag is shown in Figure 36T as SEQ ID NO:20.

[0135] Rab1a S25N The amino acid sequence of the fusion protein containing the nucleotide sequence of the nucleotide sequence of the fusion protein and an N-terminal luciferase tag is shown in Figure 36U as SEQ ID NO: 21. It is expressed in cells after transfection with either the DNA expression plasmids or mRNA described above.

[0136] mRNA treatment: Rab1a DN (also referred to as MG-008, and containing the ORF shown in Figure 36T as SEQ ID NO: 20. In this example, mRNA expressing a fusion protein containing Rab1aS25N and an N-terminal luciferase tag was used), in this example, transfection of cancer cells with mRNA expressing a fusion protein containing Rab1aS25N and an N-terminal luciferase tag was used. The protocol was performed according to the following transfection protocol: reagent: Lipofectamine™ MessengerMAX™ Transfection Reagent (Thermo Fisher Scientific) Protocol: 1. Seeding cells (5000 cells / well) 2. Incubate at 37°C for 16 hours 3. Transfection of mRNA using Lipofectamine™ MessengerMAX™ Transfection Reagent (96-well plates) (a) MessengerMAX™ Reagent diluted with Opti-MEM™ medium (each well: 5 μL Opti-MEM™ medium, 0.3 μL MessengerMAX™ Reagent) (b) Diluted MessengerMAX™ Reagent in Opti-MEM™ Medium, incubated at room temperature for 10 minutes. (c) Dilution of mRNA with Opti-MEM™ medium (each well: 5 μL of Opti-MEM™ medium, 0.1 μg of mRNA) (d) Add diluted mRNA to diluted MessengerMAX™ Reagent and incubate at room temperature for 5 minutes. (e) Remove 50 μL of cell culture medium (96-well). (f) Add mRNA-lipid complexes to cells (10 μL per well) (g) Add 50 μL of fresh culture medium to each well. 4. Incubate at 37°C for 24 hours 5. Remove Transfection Reagent-containing Medium 6. Wash twice with PBS 7. Add 100 μL of fresh culture medium to each well 8. Incubate at 37°C for 48 hours 9. Analyzing Cell Viability by Crystal Violet Staining

[0137] Several different cancer cell types were tested, as shown in the accompanying figures. For each cell type, cell viability after treatment with mRNA expressing MG-008 was compared to that of comparator control cells and to that of comparator cells treated with Lipofectamine MessengerMAX delivery vehicle (but without active MG-008, as another control comparator). For comparison, Vero cells (Chlorocereus subtilis (green monkey)) were also tested, demonstrating the selectivity of MG-008 for reducing cell viability of cancer cells compared to non-cancerous cells.

[0138] The following cancer cell lines were tested in this study: A549 cells (lung adenocarcinoma), HCT116 cells (colon carcinoma), HT29 cells (colon adenocarcinoma), HuCCT1 (cholangiocarcinoma), PC-3 cells (prostate carcinoma), RD cells (embryonic rhabdomyosarcoma), SC-M1 cells (gastric carcinoma), U-87MG cells (glioblastoma), SW480 cells (colon adenocarcinoma), PANC-1 cells (pancreatic ductal adenocarcinoma), OECM-1 cells (oral squamous cell carcinoma), OC2 cells (oral squamous cell carcinoma), and MIA cells (squamous cell carcinoma). PaCa-2 cells (pancreatic ductal adenocarcinoma), MDA-MB-468 cells (breast adenocarcinoma), MDA-MB-231 cells (breast adenocarcinoma), MCF-7 cells (invasive ductal carcinoma), Mahlavu cells (hepatocellular carcinoma), HSC-3 (squamous cell carcinoma of the tongue), HeLa cells (papillomavirus-associated endocervical adenocarcinoma), and HCT116 cells (colon carcinoma). Data are shown in the figure.

[0139] For all cancer cells tested, in this example, Rab1a S25N Rab1a DN Treatment with mRNA expressing MG-008 resulted in a significant decrease in cell viability of cancer cells compared to controls. Vero cells (Chlorocebus subtilis (green monkey)) were also tested (Figure 8), demonstrating the selectivity of MG-008 / Rab1aDN for reducing cell viability of cancer cells compared to non-cancer cells. Data for each cell type are shown in the figures. Each of these provides representative phase-contrast microscopy images of control-, transfection vehicle-, and MG-008-treated cells, along with a bar graph showing the measured % cell viability (by crystal violet staining) for each.

[0140] Treatment with DNA plasmids Rab1a DN Transfection of various different cancer cells with two different DNA plasmids expressing Rab1aS25N and an N-terminal luciferase tag (also referred to as MG-008-Luc), which contains the ORF shown in Figure 36S as SEQ ID NO: 19, was used. Rab1a without an N-terminal luciferase tag S25NA DNA plasmid expressing MG-008 (also referred to as MG-008) was also tested. Transfections were performed according to the following transfection protocol: reagent: Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific) Protocol: 1. Seeding cells (5000 cells / well) 2. Incubate at 37°C for 16 hours 3. Transfection of Plasmid DNA with Lipofectamine™ 2000 Transfection Reagent (96-well Plate) (a) Lipofectamine™ 2000 Reagent diluted with Opti-MEM™ medium (each well: 5 μL Opti-MEM™ medium, 0.3 μL Lipofectamine™ 2000 Reagent) (b) Plasmids were diluted in Opti-MEM™ medium (each well: 5 μL Opti-MEM™ medium, 0.1 μg plasmid DNA). (c) Add diluted plasmid DNA to diluted Lipofectamine™ 2000 reagent and incubate at room temperature for 5 minutes. (d) Remove 50 μL of cell culture medium (96-well). (e) Add the plasmid DNA-lipid complex to the cells (10 μL per well). (f) Add 50 μL of fresh culture medium to each well. 4. Incubate at 37°C for 24 hours 5. Remove Transfection Reagent-containing Medium 6. Wash twice with PBS 7. Add 100 μL of fresh culture medium to each well 8. Incubate at 37°C for 48 hours 9. Analyzing Cell Viability by Crystal Violet Staining

[0141] Several different cancer cell types were tested, as shown in Figures 24-40. For each cell type, cell viability after treatment with DNA plasmids expressing MG-008 or MG-008-Luc was compared to the cell viability of comparator control cells and to the cell viability of comparator cells treated with Lipofectamine 2000 delivery vehicle (but without active MG-008, as another control comparator). For comparison, Vero cells (Chlorocereus subtilis (green monkey)) were also tested, demonstrating the selectivity of MG-008 for reducing cell viability of cancer cells compared to non-cancerous cells.

[0142] The following cancer cell lines were tested in this study: U-87MG cells (glioblastoma), SW480 cells (colon adenocarcinoma), SC-M1 (gastric carcinoma), PC-3 cells (prostate carcinoma), PANC-1 cells (pancreatic ductal adenocarcinoma), OC2 cells (oral squamous cell carcinoma), MIA PaCa-2 cells (pancreatic ductal adenocarcinoma), MDA-MB-468 cells (breast adenocarcinoma), MDA-MB-231 cells (breast adenocarcinoma), MCF-7 cells (invasive ductal carcinoma), Mahlavu cells (hepatocellular carcinoma), HuCCT1 cells (cholangiocarcinoma), HT29 cells (colon adenocarcinoma), HeLa cells (papillomavirus-associated cervical adenocarcinoma), HCT116 cells (colon carcinoma), and A549 cells (lung adenocarcinoma). Data are shown in Figures 1-35.

[0143] For all cancer cells tested, in this example, Rab1a S25N Rab1a (either with or without an N-terminal luciferase tag) DNTreatment with a DNA plasmid expressing MG-008 / Rab1a resulted in a significant decrease in cell viability of cancer cells compared to controls. Vero cells (Chlorocebus subtilis (green monkey)) were also tested, demonstrating the selectivity of MG-008 / Rab1aDN for reducing cell viability of cancer cells compared to non-cancer cells. Data for each cell type are shown in Figures 1-35. Each of these provides representative phase-contrast microscopy images of cells treated with control, transfection vehicle, MG-008, or MG-008-Luc, along with a bar graph showing the measured % cell viability (by crystal violet staining).

[0144] The data from these studies demonstrate that Rab1a is highly potent against a wide variety of different cancer cell types. DN supports the anti-cancer effects of Rab1a. DN (in this example, Rab1a S25N Rab1a DN was introduced into cells either as transfected mRNA nucleic acid or transfected DNA expression plasmid. The latter was shown to inhibit Rab1a expression in cancer cells. S25N , or Rab1a S25N A fusion protein containing an N-terminal luciferase tag can be expressed. Results show selectivity for reducing cell viability in cancer cells compared to normal cells. Anticancer effects were observed in many different cancer cell types, supporting broad anticancer applicability.

[0145] One or more exemplary embodiments have been described by way of example, and it will be understood by those skilled in the art that numerous variations and modifications can be made without departing from the scope of the invention, as defined in the claims.

[0146] Example 2: Rab1a encapsulated in lipid nanoparticles DN In vivo reduction of tumor cell growth and metastasis following treatment with A series of experiments were conducted to investigate the in vivo anti-cancer effects of F11. F11, which is referred to in the following paragraphs and Figures 37-40, was developed to inhibit Rab1a encapsulated in lipid nanoparticles (LNPs). DN is.

[0147] In this example, the efficacy of F11 treatment in Balb / c mice injected with 4T1 cancer cells was demonstrated. Each female Balb / c mouse (7 weeks old) received 1 × 10 6 4T1-Fluc-Neo cells (a luciferase-expressing mouse breast cancer cell line) were injected into the untreated control mice. One week later, the mice received a single intravenous (IV) dose of 0.05 mpk F11 (in 100 μl of PBS). Untreated control mice were injected with PBS alone. Subsequently, in vivo imaging system (IVIS) images of bioluminescence from the 4T1 tumor cells were recorded weekly for the next 7 consecutive weeks (Figure 37). The total luciferin flux obtained from this experiment was then quantified (Figure 38). Metastasis was observed in the untreated control mice on day 30 (Figure 38A), and the mice died on day 37. No metastasis was observed in the F11-injected mice throughout the entire 7-week experiment (Figure 38B). These data suggest that a single IV treatment with F11 could effectively suppress 4T1 metastasis in this mouse model.

[0148] Further experiments were performed to confirm the morphological changes occurring at the FT1 injection site in Balb / c mice after administration of F11 or PBS (control). Again, as described above, each female Balb / c mouse was injected with 4T1 cancer cells into the left inguinal fourth mammary gland. One week later, the mice received a single intravenous (IV) dose of F11, while control mice were injected with PBS alone. Morphological changes at the 4T1 injection site were monitored. In control mice, tumor masses were palpable on day 14 after IV treatment, which continued to grow until the mice died on day 35 (image in the upper panel of Figure 39). In F11-treated mice, tumor masses were undetectable until day 35 after IV treatment (image in the lower panel of Figure 39). These data suggest that a single F11 treatment could effectively attenuate 4T1 tumor cell growth in mice.

[0149] To supplement the treatment efficacy data, mouse weights were monitored to determine whether there were any adverse health effects from F11 treatment. In this experiment, Balb / c mice were injected with 4T1 tumor cells as described above. One week later (day 0), the mice received a single intravenous (IV) dose of F11 or PBS as a control. Body weights were measured until the end of the experiment (a total of 7 weeks after IV treatment). The weight of the PBS control mice did not change significantly before they died on day 35 (blue line, lower line). F11-treated mice showed weight gain throughout the experiment (orange line, upper line). These data suggest that F11 treatment did not exert any adverse effects on the overall health of this mouse model.

[0150] References Li, W.-W., Li, J., and Bao, J.-K., "Microautophagy: lesser-known self-eating," Cellular and Molecular Life Sciences, Vol. 69, pp. 1125-1136 (2011). Fujiwara, Y. et al., "Direct uptake and degradation of DNA by lysosomes." - PubMed - NCBI. Autophagy, Vol. 9, pp. 1167-1171 (2014). Andrews, NW, "Lysosomes and the plasma membrane," Journal of Cell Biology, 158, 389-394 (2002). Hofmann, I. and Munro, S., "An N-terminally acetylated Arf-like GTPase is localized to lysosomes and affects their motility," Journal of Cell Science, 119, 1494-1503 (2006). Fraldi, A. et al., "Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders," The EMBO Journal, Vol. 29, pp. 3607-3620 (2010). Pankiv, S. et al., "FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport," Journal of Cell Biology, 188, 253-269 (2010). R. and Bonifacino, J.S., "Lysosome Positioning Influences mTORC2 and AKT Signaling," Molecular Cell, 75, 26-38, e3 (2019). Pous, C. and Codogno, P., "Lysosome positioning coordinates mTORC1 activity and autophagy," Nature Cell Biology, 13, 342–344 (2011). Cabukusta, B. and Neefjes, J., "Mechanisms of lysosomal positioning and movement," Traffic, Vol. 19, pp. 761-769 (2018). Jia, R. and Bonifacino, J.S., "Lysosome Positioning Influences mTORC2 and AKT Signaling," Molecular Cell, Vol. 75, pp. 26-38, e3 (2019). Pu, J., Guardia, C.M., Keren-Kaplan, T., and Bonifacino, J.S., "Mechanisms and functions of lysosome positioning," Journal of Cell Science, Vol. 129, pp. 4329-4339 (2016). Katherine R. Parzych, DJK, “An Overview of Autophagy: Morphology, Mechanism, and Regulation,” Antioxidants & Redox Signaling, Vol. 20, pp. 460–473 (2014). Rabanal-Ruiz, Y., and Korolchuk, V.I., “mTORC1 and Nutrient Homeostasis: The Central Role of the Lysosome,” International Journal of Molecular Sciences, Vol. 19, p. 818 (2018). Jia, R. and Bonifacino, J.S., "Lysosome Positioning Influences mTORC2 and AKT Signaling," Molecular Cell, Vol. 75, pp. 26-38, e3 (2019). Allyson L. Anding, EHB, "Cleaning House: Selective Autophagy of Organelles," Developmental Cell, Vol. 41, pp. 10-22 (2017) De Bortoli, M. et al., "Lipid accumulation in human breast cancer cells injured by iron depletors," Undefined, Vol. 37, p. 1 (2018) Ipsen, D.H., Lykkesfeldt, J., and Tveden-Nyborg, P., "Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease," Cellular and Molecular Life Sciences, Vol. 75, pp. 3313-3327 (2018). Nakagawa, H. et al., "Lipid Metabolic Reprogramming in Hepatocellular Carcinoma," Cancers, Vol. 10, p. 447 (2018) Namba, Y. et al., "Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells," Frontiers in Oncology, Vol. 8, e0191109 (2018) Shyu, P., Wong, X.F.A., Crasta, K., and Thibault, G., “Dropping in on lipid droplets: insights into cellular stress and cancer,” Bioscience Reports, Vol. 38, BSR20180764 (2018). Christos E. Zois, ALH, "Glycogen metabolism has a key role in the cancer microenvironment and provides new targets for cancer therapy," Journal of Molecular Medicine, Vol. 94, pp. 137-154 (2016) Zois, C.E., Favaro, E., and Harris, A.L., "Glycogen metabolism in cancer," Biochemical Pharmacology, Vol. 92, pp. 3-11 (2014). Parachoniak CA, Park M. "Dynamics of receptor trafficking in tumorigenicity." Trends in Cell Biology, May 2012; 22(5): 231-40. Anderson RG, Ghiraldeli LP, Pardee TS. "Mitochondria in cancer metabolism, an or-ganelle whose time has come?" Biochimica et Biophysica Acta: Reviews on Cancer. August 2018; 1870(1):96-102. Davidson SM, Vander Heiden MG, "Critical Functions of the Lysosome in Cancer Biology," Annual Review of Pharmacology and Toxicology, January 6, 2017; Vol. 57: pp. 481-507. Zhitomirsky B, Assaraf YG, "Lysosomes as mediators of drug resistance in cancer," Drug Resistance Updates, January 2016; Vol. 24: pp. 23-33 Rudzinska M, Parodi A, Soond SM, Vinarov AZ, Korolev DO, Morozov AO, Daglioglu C, Tutar Y, Zamyatnin AA Jr. "The Role of Cysteine ​​Cathepsins in Cancer Progression and Drug Resistance." International Journal of Molecular Sciences, July 23, 2019; 20(14): 3602. Kim KH, Lee MS, "Autophagy - a key player in cellular and body metabolism," Nature Reviews Endocrinology, June 2014; Vol. 10(6): pp. 322-37 Parzych KR, Klionsky DJ, "An overview of autophagy: morphology, mechanism, and regulation." Antioxidants & Redox Signaling, 2014, January 20; 20(3): 460-73. Gong C, Bauvy C, Tonelli G, Yue W, Delomenie C, Nicolas V, Zhu Y, Domergue V, Marin-Esteban V, Tharinger H, Delbos L, Gary-Gouy H, Morel AP, Ghavami S, Song E, Codogno P, Mehrpour M. Beclin 1 and autophagy are required for the tumorigenicity of breast cancer stem-like / progenitor cells. Oncogene 2013 May 2; 32(18): pp. 2261-72, 2272e.1-11 Mathiassen SG, De Zio D, Cecconi F. "Autophagy and the Cell Cycle: A Complex Landscape." Frontiers in Oncology, March 31, 2017; Vol. 7: 51. Simon HU, Friis R. "ATG5: a distinct role in the nucleus." Autophagy 2014 January; 10(1): 176-7 Gozuacik D, Kimchi A. "Autophagy as a cell death and tumor suppressor mechanism." Oncogene 2004 April 12; 23(16): 2891-906. Pathania AS, Guru SK, Kumar S, Kumar A, Ahmad M, Bhushan S, Sharma PR, Mahajan P, Shah BA, Sharma S, Nargotra A, Vishwakarma R, Korkaya H, Malik F. "Interplay between cell cycle and autophagy induced by boswellic acid analog." Scientific Reports, September 29, 2016; Vol. 6: p. 33146. Fuchs Y, Steller H. "Programmed cell death in animal development and disease." Cell 2011, November 11; 147(4): 742-58 Kroemer G, Levine B. "Autophagic cell death: the story of a misnomer." Nature Reviews Molecular Cell Biology, December 2008; 9(12): 1004-10. Rosebeck S, Alonge MM, Kandarpa M, Mayampurath A, Volchenboum SL, Jasielec J, Dytfeld D, Maxwell SP, Kraftson SJ, McCauley D, Shacham S, Kauffman M, Jakubowiak AJ. Synergistic Myeloma Cell Death via Novel Intracellular Activation of Caspase-10-Dependent Apoptosis by Carfilzomib and Selinexor. Selinexor,” Molecular Cancer Therapeutics, January 2016; Vol. 15(1): pp. 60-71 Scherz-Shouval R, Weidberg H, Gonen C, Wilder S, Elazar Z, Oren M. "p53-dependent regulation of autophagy protein LC3 supports cancer cell survival under prolonged starvation." Proceedings of the National Academy of Sciences of the United States of America, October 26, 2010; 107(43): 18511-6. Filomeni G, De Zio D, Cecconi F. "Oxidative stress and autophagy: the clash between damage and metabolic needs." Cell Death & Differentiation, March 2015; 22(3): 377-88. Liou GY, Storz P, "Reactive oxygen species in cancer," Free Radical Research, May 2010; Vol. 44(5): pp. 479-96 Auten, RL, Davis, JM. "Oxygen toxicity and reactive oxygen species: the devil is in the details." Pediatric Research, August 2009; 66(2): 121-7. Perillo B, Di Donato M, Pezone A, Di Zazzo E, Giovannelli P, Galasso G, Castoria G, Migliaccio A. "ROS in cancer therapy: the bright side of the moon." Experimental & Molecular Medicine, February 2020; 52(2): 192-203. Martinez-Lopez N, Athonvarangkul D, Singh R. "Autophagy and aging." Advances in Experimental Medicine and Biology 2015; 847: 73-87 Tan MH, Mester L, Ngeow J, Rybicki LA, Orloff MS, Eng C. "Lifetime cancer risks in individuals with germline PTEN mutations." Clinical Cancer Research 2012;15 Jan;18(2):400-7. Liu EY, Xu N, O'Prey J, Lao LY, Joshi S, Long JS, O'Prey M, Croft DR, Beaumatin F, Baudot AD, Mrschtik M, Rosenfeldt M, Zhang Y, Gillespie DA, Ryan KM. "Loss of autophagy causes a synthetic lethal deficiency in DNA repair." Proceedings of the National Academy of Sciences of the United States of America, January 20, 2015; 112(3): 773-8. WO2017 / 008141 - Lysosomal Degradation of Lipids and Proteins and Method of Use Thereof

[0151] All references cited in and throughout this application are hereby incorporated by reference in their entirety.

Images