Compositions and methods for enhancing the fitness of tumor-infiltrating natural killer cells

Abstract

Disclosed herein are compositions and methods for increasing the viability of natural killer (NK) cells. NK cells may be modified to increase the expression of CaMKK2. The modified NM cells may be administered to a subject to treat cancer.

Description

Docket No.028193-0031-WO01 / 7879 COMPOSITIONS AND METHODS FOR ENHANCING THE FITNESS OF TUMOR-INFILTRATING NATURAL KILLER CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 454,999, filed March 28, 2023, the entire contents of which are hereby incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant 1F30CA261005 awarded by the National Cancer Institute (NCI) and grant W81XWH-20-1-0497 awarded by the Department of Defense (DOD). The government has certain rights in the invention. FIELD

[0003] This disclosure relates to natural killer (NK) cells and increasing their survival for the treatment of cancer. INTRODUCTION

[0004] Solid tumors create unique metabolic challenges that tumor-associated cells must navigate to survive and proliferate. Calcium / calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a calcium-activated serine / threonine kinase that regulates metabolic functions through several pathways, including the AMP-activated protein kinase (AMPK) and calcium / calmodulin-dependent protein kinase type IV (CaMKIV) pathways. Originally studied in the context of central nervous system physiology, CaMKK2 was subsequently found to play important regulatory roles in many tissues, most notably control of gluconeogenesis in the liver, insulin signaling in the pancreas, and osteoblast activity in the bone. Dysregulated CaMKK2 signaling also contributes to the pathology of several diseases, including nonalcoholic fatty liver disease (NAFLD) and tumor progression in many different cancers. Ectopic expression of CaMKK2 results in the suppression of anoikis, enhanced migratory capacity, and increased proliferation of cancer cells.

[0005] In the immune system, CaMKK2 is highly expressed in early hematopoietic stem and progenitor cells, where it promotes quiescence and suppresses differentiation. CaMKK2 expression in granulocyte-monocyte progenitor cells promotes monocyte lineage commitment, and its expression is attenuated as granulocytes mature. However, CaMKK2 remains highly expressed in monocytes and monocyte-derived macrophages, and deletionDocket No.028193-0031-WO01 / 7879 of this kinase attenuates macrophage-mediated inÀammatory responses. Further, the role of CaMKK2 in other tumor-in¿ltrating immune populations has not been studied, limiting the development of a complete model of how CaMKK2 inÀuences tumor pathobiology.

[0006] Given its role in promoting tumor progression, there is interest in developing CaMKK2 inhibitors as anticancer therapeutics. The most widely used CaMKK2 antagonist, the competitive inhibitor STO-609, has shown ef¿cacy in mouse models of breast cancer and NAFLD. However, the poor pharmaceutical properties of this drug coupled with its established off-target activities have been an impediment to its clinical development. Several new inhibitors are emerging with improved properties, and these new drugs are divided into two functional categories: ATP-competitive inhibitors of kinase function, and ligand-directed degraders (LDD) of the enzyme. LDDs are a new class of bi-speci¿c molecules that feature a target-binding ligand paired with a ubiquitin ligase-recruitment domain that promotes proteasomal degradation of the target protein. LDDs may offer advantages over kinase inhibitors, including activity against inhibitor-resistant cancers and enhanced target selectivity, although it remains to be determined whether CaMKK2-speci¿c LDDs and competitive inhibitors are functionally equivalent. As these compounds are currently under investigation as anticancer therapeutics, there remains a need to better understand the effects of CaMKK2 modulation on immune cell function. SUMMARY

[0007] In an aspect, the disclosure relates to a method of increasing survival and / or proliferation of a natural killer (NK) cell in a tumor of a subject. The method may include isolating the natural killer (NK) cell from a biological sample from the subject to generate an isolated NK cell; administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell; and administering to the subject the modified NK cell.

[0008] In a further aspect, the disclosure relates to a method of increasing the level of CaMKK2 in a natural killer (NK) cell in a tumor of a subject. The method may include isolating the natural killer (NK) cell from a biological sample from the subject to generate an isolated NK cell; administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell; and administering to the subject the modified NK cell.

[0009] Another aspect of the disclosure provides a method of treating cancer in a subject. The method may include isolating a natural killer (NK) cell from a biological sampleDocket No.028193-0031-WO01 / 7879 from the subject to generate an isolated NK cell; administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell; and administering to the subject the modified NK cell.

[0010] In some embodiments, the modified NK cell has reduced rate of apoptosis compared to a control NK cell. In some embodiments, the modified NK cell has an increased rate of replication and / or growth compared to a control NK cell. In some embodiments, the CaMKK2 expressed in the modified NK cell maintains scaffolding activity. In some embodiments, the modified NK cell constitutively expresses a level of CaMKK2 greater than that of a control NK cell. In some embodiments, the modified NK cell expresses a level of CaMKK2 that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1.5-fold, 2-fold, 2.5-fold, 3- fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold greater than that of a control NK cell. In some embodiments, the administration of the modified NK cell decreases the rate of progression of tumor growth in the subject, as compared to the rate of progression of tumor growth prior to the modified NK cell treatment. In some embodiments, the administration of the modified NK cell decreases the rate of cancer metastasis in the subject, as compared to the rate of cancer metastasis prior to the modified NK cell treatment. In some embodiments, the administration of the modified NK cell decreases the size of a tumor in the subject, as compared to the size of the tumor prior to the modified NK cell treatment. In some embodiments, the method further comprises administering to the isolated NK cell a polynucleotide encoding LDH to increase the level of CaMKK2 in the isolated NK cell. In some embodiments, the LDH comprises a polypeptide having the amino acid sequence of SEQ ID NO: 4, and / or wherein the polynucleotide encoding LDH comprises the sequence of SEQ ID NO: 5 or 6. In some embodiments, a vector comprises the polynucleotide encoding CaMKK2. In some embodiments, the vector is an AAV vector. In some embodiments, the polynucleotide encoding CaMKK2 comprises the sequence of SEQ ID NO: 2 or 3, and / or wherein the CaMKK2 comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified NK cell is administered to the subject once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months. In some embodiments, the modified NK cell is administered to the subject for 1 year, 2 years, 3 years, 4 years, 5 years, or moreDocket No.028193-0031-WO01 / 7879 than 5 years. In some embodiments, the method further comprises administering to the subject at least one additional therapy. In some embodiments, the at least one additional therapy is selected from chemotherapy, immunotherapy, radiation therapy, hormone therapy, targeted drug therapy, cryoablation, and surgery, or a combination thereof. In some embodiments, the chemotherapy is selected from an antimitotic agent, an alkylating agent, an antimetabolite, an antimicrotubule agent, a topoisomerase inhibitor, a cytotoxic agent, a cell cycle inhibitor, a growth factor inhibitor, a histone deacetylase (HDAC) inhibitor, and an inhibitor of a pathway that cross-talks with and activates ER transcriptional activity, or a combination thereof. In some embodiments, the subject has a solid cancer. In some embodiments, the solid cancer comprises melanoma, colon cancer, breast cancer, lung cancer, head and neck cancer, laryngeal cancer, oral cancer, esophageal cancer, gastric cancer, stomach cancer, colon cancer, bladder cancer, liver cancer, pancreatic cancer, kidney cancer, brain cancer, bone cancer, glioblastoma, sarcoma, ovarian cancer, endometrial cancer, or prostate cancer, or a combination thereof.

[0011] Another aspect of the disclosure provides a composition for increasing survival and / or proliferation of a natural killer (NK) cell, the composition comprising a polynucleotide encoding CaMKK2 to express a level of CaMKK2 in the NK cell greater than that of a control NK cell. In some embodiments, the polynucleotide encoding CaMKK2 comprises the sequence of SEQ ID NO: 4 or 5.

[0012] The disclosure provides for other aspects and embodiments that will be apparent in light of the following detailed description and accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG.1 schematically shows key metabolic regulatory pathways in natural killer (NK) cells.

[0014] FIGS.2A-2C show that calcium / calmodulin-dependent protein kinase kinase 2 (CaMKK2) is expressed in CD11b+NK cells and double-negative thymocytes. FIG.2A graphically shows flow cytometry analysis data where splenic immune cells from healthy CaMKK2-eGFP reporter mice were harvested and analyzed for enhanced green fluorescent protein (eGFP) fluorescence using flow cytometry. Each distribution represents >500 cells. The eGFP median fluorescence intensity (MFI) among the eGFP+subset of monocytes and the eGFP+subset of CD11b+NK cells (see depicted cutoff line) was compared to the eGFP MFI in all CD4 T-cells. N=2, standard error is shown. FIG.2B graphically shows flow cytometry analysis data where mature NK cells (NK1.1+ / CD11b+) were isolated from variousDocket No.028193-0031-WO01 / 7879 tissues of healthy CaMKK2-eGFP reporter mice and analyzed for eGFP fluorescence using flow cytometry. Each distribution represents >1000 cells. The percentage of CD11b+NK cells that are eGFP+(see depicted cutoff line) was compared across tissues. N=2, standard error is shown. FIG.2C graphically shows flow cytometry analysis data where thymocytes were harvested from healthy CaMKK2-eGFP reporter mice and analyzed for eGFP fluorescence using flow cytometry. The double negative thymocyte fraction (CD3í / CD4í / CD8í) was divided into eGFP+and eGFPísubpopulations, which were further analyzed for CD44 expression. Each distribution represents >250 cells; data is representative of 2 mice.

[0015] FIGS.3A-3B show that NK cells are the only cells from the lymphoid lineage that express CaMKK2. FIG.3A graphically shows flow cytometry analysis data where various tissues were harvested from healthy CaMKK2-eGFP reporter mice, and several immune subsets were stained for flow cytometry analysis of eGFP fluorescence. Each distribution represents >500 cells; data is representative of two mice. FIG.3B shows the flow cytometry gating strategy for data shown in FIG.2A.

[0016] FIG.4A-4H show that CaMKK2 expression is upregulated in tumor-infiltrating NK cells. FIG.4A graphically shows flow cytometry analysis data where splenic NK cells were harvested from CaMKK2-eGFP reporter mice, cultured in 50% EO771 tumor-conditioned media (TCM) for 24-72 hours, and analyzed for eGFP fluorescence via flow cytometry. FIG. 4B-4C show eGFP fluorescence analysis of fourteen-day subcutaneous EO771 tumors that were harvested from two CaMKK2-eGFP reporter mice along with spleens. NK cells from tumor and spleen (FIG.4C), or NK cells, T-cells, and macrophages from the tumor (FIG.4D) were analyzed for eGFP fluorescence. FIG.4E shows eGFP fluorescence analysis of splenic NK cells that were harvested from CaMKK2-eGFP reporter mice bearing fourteen- day subcutaneous EO771 tumors and from healthy control reporter mice. FIG.4E-4F show eGFP expression analysis for a variety of NK cell subsets from fourteen-day subcutaneous EO771 tumors that were harvested from CaMKK2-eGFP reporter mice. Each distribution represents >1000 cells; data is representative of 2 mice. FIG.4G shows a Western immunoblot for bone marrow-derived macrophages (BMDMs), splenic T-cells, and splenic NK cells harvested from wild type (WT) and CaMKK2 knock out (KO) BL / 6 female mice that were cultured in 50% EO771 tumor-conditioned media (TCM) or normal growth media (NM) for 72 hours. CaMKK2 and the ȕ-actin control were measured via Western immunoblotting on a 7.5% acrylamide gel. FIG.4H depicts the flow cytometry gating strategy for FIGS.4A-Docket No.028193-0031-WO01 / 7879

[0017] FIGS.5A-5H show that CaMKK2 ablation reduces NK cell viability. FIGS.5A-5C graphically show flow cytometry analysis data for splenic NK cells that were isolated from WT or CaMKK2 KO BL / 6 females and cultured for 96 hours in 50% EO771 TCM or normal growth media (NM). Flow cytometry was used to determine, the total number of live NK cells as a proportion of the dimethyl sulfoxide (DMSO) control (FIG.5A), the viability of the NK cells (FIG.5B), and Annexin V staining on the NK cells (FIG.5C). FIG.5D shows a Western immunoblot for splenic NK cells that were harvested from WT BL / 6 females and cultured for 72 hours in 50% EO771 TCM containing 2.5 μM CC3240 or DMSO vehicle. CaMKK2 and the ȕ-actin control were measured via Western immunoblotting on a 7.5% acrylamide gel. FIGS.5E-5G graphically show flow cytometry analysis data for splenic NK cells that were isolated from WT BL / 6 females and cultured for 72 hours in 50% EO771 TCM or normal growth media (NM) containing 2.5 μM CC3240 or DMSO vehicle. Flow cytometry was used to determine the total number of live NK cells as a proportion of the DMSO control (FIG.5E), the viability of the NK cells (FIG.5F), and Annexin V staining on the NK cells (FIG.5G). FIG.5H graphically shows flow cytometry analysis data for splenic NK cells that were isolated from WT BL / 6 females and cultured for 72 hours in 50% A7C11 TCM containing 2.5 μM CC3240 or DMSO vehicle. Flow cytometry was used to determine NK cell viability. For all panels: N=3, and standard error is shown.

[0018] FIGS.6A-6E show that CaMKK2 ablation reduces NK cell proliferation and migration. FIG.6A graphically shows flow cytometry analysis data for splenic NK cells that were isolated from WT or CaMKK2 KO BL / 6 females and cultured for 96 hours in 50% EO771 TCM or normal growth media (NM). Flow cytometry was used to determine Ki67 staining. N=3, standard error shown. FIG.6B graphically shows flow cytometry analysis data for splenic NK cells that were isolated from WT BL / 6 females, stained with CFSE, and cultured for 72 hours in 50% EO771 TCM or NM containing 2.5 μM CC3240 or DMSO vehicle. Flow cytometry was used to measure dilution of the carboxyfluorescein succinimidyl ester (CFSE) dye. N=3, standard error shown. FIG.6C graphically shows flow cytometry analysis data for splenic NK cells that were isolated from WT BL / 6 females and cultured for 72 hours in 50% EO771 TCM containing 2.5 μM CC3240 or DMSO vehicle. 500k live NK cells were transferred to the upper chambers of a migration plate, separated from Chemokine ligand 4 (CCL4)-containing media by a barrier with 5 μm pores. After 3 hours, cell number in the bottom chamber was measured via flow cytometry. N=3, standard error shown. FIG.6D graphically shows flow cytometry analysis data for splenic NK cells that were harvested from WT or CaMKK2 KO BL / 6 females and cultured for 96 hours in normal growth media containing 100 ng / mL Interleukin 2 (IL2) and Interleukin 15 (IL15). C-CDocket No.028193-0031-WO01 / 7879 chemokine receptor type 5 (CCR5) expression was measured via flow cytometry and compared to an isotype control. FIG.6E shows the flow cytometry gating strategy.

[0019] FIGS.7A-7C show that CaMKK2 ablation does not affect NK cell cytotoxic potency. FIGS.7A-7B graphically show flow cytometry analysis data for splenic NK cells that were isolated from WT BL / 6 females and cultured for 72 hours in 50% EO771 TCM containing 2.5 ^M CC3240 or DMSO vehicle. FIG.7A graphically shows flow cytometry analysis data where the median fluorescence intensity (MFI) of key functional markers was measured using flow cytometry. N=2, standard error shown. FIG.7B graphically shows flow cytometry analysis data where live NK cells were co-cultured with CFSE-stained YAC-1 cells at the stated ratios for 4 hours, and YAC-1 viability was measured using flow cytometry. YAC-1 cells were also cultured alone (YAC-1 alone) or with 25% ethanol (YAC1 + EtOH). N=3, standard error is shown. FIG.7C graphically shows flow cytometry analysis data for splenic NK cells that were harvested from WT or CaMKK2 KO mice and cultured in 50% EO771 media for 96 hours. Live NK cells were co-cultured with CFSE-stained YAC-1 cells at a 1:5 ratio for 4 hours, and YAC-1 viability was measured via flow cytometry. N=3, standard error is shown.

[0020] FIGS. 8A-8G show that CaMKK2 ligand-directed degraders (LDDs), but not competitive inhibitors, reduce NK cell fitness. FIGS.8A-8B show splenic NK cells isolated from WT BL / 6 females were cultured for 96 hours in 50% EO771 TCM or normal growth media (NM) containing DMSO vehicle, the LDD CC3240 or CC3756 (1 μM), or the competitive inhibitor GSKi, YL36, or CC8977 (1 μM). FIG.8A graphically shows NK cell viability. FIG.8B graphically shows expression of Ki67 measured using flow cytometry. N=3, standard error shown. FIG.8C shows Western immunoblot analysis results for splenic NK cells isolated from WT BL / 6 females were cultured for 96 hours in normal media containing 10 mM lactic acid and either the DMSO vehicle, the LDD CC3240 or CC3756 (1 μM), or the competitive inhibitor GSKi, YL36, or CC8977 (1 μM). A Western immunoblot was then used to analyze expression of CaMKK2, AMPKĮ, phospho-Thr172-AMPKĮ (pAMPK), and ȕ-actin (7.5% acrylamide gel, 45 ^g protein loaded per well). FIG.8D shows splenic NK cells were isolated from WT BL / 6 females and cultured for 72 hours in 50% EO771 TCM or normal growth media containing 10 ^M STO609 or DMSO vehicle. NK cell viability was measured via flow cytometry. N=2, standard error shown. FIGS.8E-8G graphically show graphically flow cytometry analysis results for splenic NK cells isolated from WT BL / 6 females that were cultured for 96 hours in 50% EO771 TCM containing DMSO vehicle, the LDD CC3240 or CC3756 (1 μM), or the competitive inhibitor GSKi, YL36, or CC8977 (1 μM). (D) CaMKK2, AMPK, pAMPK (Thr172), and ȕ-actin expression wereDocket No.028193-0031-WO01 / 7879 measured by Western immunoblotting following resolution of proteins on a 7.5% acrylamide gel. FIG.8E graphically shows MitoTracker Green FM staining, as measured via flow cytometry to determine total mitochondria mass. FIG.8F graphically shows MitoMark Red I staining, as measured via flow cytometry, to determine the mass of polarized mitochondria. N=3, standard error shown. FIG.8G graphically shows flow cytometry analysis data for splenic NK cells that were harvested from WT or KO BL / 6 females, cultured for 96 hours in 50% EO771 TCM, stained with the MitoSox ROS dye, and analyzed using flow cytometry. N=2, standard error is shown.

[0021] FIGS.9A-9B show the profile of NK cells and tumor cells used in metastasis studies. FIG.9A graphically shows flow cytometry analysis data for splenic NK cells that were harvested from WT and CaMKK2 KO BL / 6 females and cultured for 24 hours in normal media containing 100 ng / mL IL15. Various functional markers were characterized via flow cytometry. The prevalence of immature NK cells (CD27+ / CD11bí), mature 1 NK cells (CD27+ / CD11b+), and mature 2 NK cells (CD27- / CD11b+) was also measured. N=4, standard error shown. FIG.9B graphically shows flow cytometry analysis data for ȕ2mí / íEO771 cells, B16-F10 cells, and B16-F10 cells that were treated with 100 ng / mL IFNȖ for 48 hours, then analyzed for MHC Class I expression via flow cytometry. Fluorescence was compared to EO771 parent cells stained with an isotype control antibody.

[0022] FIGS.10A-10C show that NK cells from a CaMKK2 KO background demonstrate reduced activity against EO771 metastases. FIG.10A schematically illustrates that splenic NK cells from WT or CaMKK2 KO BL / 6 females were isolated, cultured in media containing IL15 for 24 hours, and intravenously transfused into NOD.Cg-Prkdc^ಠƍ^ Il2rg^ஆ¹^^^ / SzJ (NSG) mice. Two days later, luciferase-expressing ȕ2mí / íEO771 breast tumor cells were also transfused. A positive control group received only tumor cells and no NK cells. FIG.10B graphically shows whole-body metastatic burden in the EO771 study, which was measured using luciferin bioluminescence imaging at days 11 and 14 post-tumor injection. Camera exposure time was 3 minutes. N=5-6, standard error is shown. FIG.10C shows raw bioluminescence data from the day 14 imaging session.

[0023] FIGS.11A-11E show that NK cells from a CaMKK2 KO background demonstrate reduced activity against B16-F10 metastases. FIG.11A graphically shows bioluminescence results for NSG mice that were engrafted with NK cells from WT or KO mice and injected with luciferase-expressing B16-F10 tumor cells. Whole-body metastatic burden was measured using luciferin bioluminescence imaging. Results were collected 12- and 15-days post-injection. Camera exposure time was two minutes. N=7-8, standard error shown. FIG. 11B shows raw bioluminescence data from Day 15. FIGS.11C-11D graphically show flowDocket No.028193-0031-WO01 / 7879 cytometry analysis data for NK cells that were recovered post-mortem from the lungs of each mouse and analyzed using flow cytometry. FIG.11C graphically shows live NK cells as a portion of CD45+cells (NK prevalence), total live NK cells as a fraction of live tumor cells (NK count), and the fraction of all NK cells that are alive (NK viability) were measured. FIG. 11D graphically shows median fluorescence intensity (MFI) of NKp46 and Ly49c on NK cells was measured. The distribution of immature, Mature 1 NK cells, and Mature 2 NK cells was measured. N=7-8, standard error shown. FIG.11E shows the flow cytometry gating strategy.

[0024] FIGS.12A-12F demonstrate that NK cells from an inducible CaMKK2 KO background show reduced activity against B16-F10 metastases. FIG.12A schematically illustrates that NK cells were harvested from the spleens of tamoxifen-treated CaMKK2fl / fl:ERT2+mice or CaMKK2fl / fl:ERT2ímice and co-injected into the tail veins of NSG mice along with luciferase-expressing GFP+B16-F10 tumor cells. The CaMKK2fl / fl:ERT2+mice express a fusion cre-ERT2 protein that translocated to the nucleus upon tamoxifen binding, excising the floxed CaMKK2 alleles. FIG.12B shows a Western immunoblot on a 7.5% acrylamide gel, where after one week of tamoxifen dosing, BMDMs were recovered from the ERT2+mice and ERT2ínegative controls to confirm CaMKK2 ablation. FIG.12B shows whole-body luciferin bioluminescence that was measured at days 13 and 17 post- tumor cell injection. Camera exposure time was 2 minutes. N=6, standard error shown. FIG.12D shows the raw bioluminescence data from day 17. FIG.12E graphically shows flow cytometry analysis data for GFP+tumor cells that were harvested from the lungs of each mouse 17 days post-injection, then analyzed for MHC Class I expression using flow cytometry. N=6, standard error shown. FIG.12F graphically shows flow cytometry analysis data for the NK cells that were harvested from lungs of each mouse 17 days post-injection. Flow cytometry was used to measure the prevalence of NK cells among live CD45+cells (prevalence), the number of live NK cells as a proportion of live tumor cells (count), the percentage of all NK cells that are alive (viability), and Ki67 expression among NK cells (Ki67).

[0025] FIGS.13A-13D show that NK cell-specific CaMKK2 ablation produces increased metastatic progression in immune-competent mice. FIG.13A schematically illustrates the experimental method where mice were bred that expressed Cre under the Ncr1 (NKp46) promoter and contained either CaMKK2fl / fl(fl / fl) alleles or CaMKK2wt / wtalleles, and luciferase- expressing B16-F10 tumor cells were injected via tail vein into these two groups. FIG.13B shows whole-body luciferin bioluminescence measured at days 16 and 19 post-tumor injection. Camera exposure time was 3 minutes. N=11, standard error shown. FIG.13CDocket No.028193-0031-WO01 / 7879 shows raw bioluminescence data from day 19. FIG.13D graphically shows flow cytometry analysis data for NK cells that were harvested from the lungs of each mouse 19 days post- injection. Flow cytometry was used to measure the absolute number of live NK cells and the prevalence of various other immune populations within the lungs. M1 macrophages were defined as MHC Class II+ / CD206í, and M2 macrophages were defined as MHC Class IIí / CD206+. N=11, standard error is shown.

[0026] FIGS.14A-14C shows that small molecule tumor factor(s) promotes CaMKK2 upregulation. FIG.14A graphically shows flow cytometry results for splenic NK cells from WT BL / 6 females that were cultured in 50% E0771 TCM (Normal) or 50% E0771 TCM that had been incubated at 56 degrees for 30 minutes (Heat-inactivated). Cells were treated with either 2.5 ^M CC3240 or DMSO vehicle for 72 hours, and total number of live NK cells was measured via flow cytometry. N=2, standard error shown. FIG.14B shows A7C11 TCM, E0771 TCM, and normal growth media (control) analysis via mass spectrometry for 217 common metabolites. Twelve metabolites, highlighted at the top of the chart, were significantly elevated by a factor of eight or more in both A7C11 TCM and E0771 TCM compared to the normal media (NM) control. N=3, standard error shown. FIG.14C graphically shows the relative levels of two metabolites known to influence tumor immunity are shown. N=3, standard error shown.

[0027] FIGS.15A-15E show that lactic acid exposure is sufficient to mediate CaMKK2 upregulation in NK cells. FIGS.15A-15B graphically show flow cytometry analysis data for splenic NK cells that were isolated from CaMKK2-eGFP reporter mice and cultured for 24-96 hours in normal growth media (NM), 50% EO771 tumor-conditioned media (TCM), or normal media with 10 mM lactic acid (LA). Flow cytometry was used to measure the level of eGFP expression relative to the NM group at 24 hours in culture (FIG.15A), or 96 hours in culture (FIG.15B). The raw eGFP fluorescence peaks at 96 hours are also shown. N=3, standard error shown. FIG.15C shows Western immunoblot analysis for mixed splenocytes, pure splenic NK cells, and bone marrow-derived macrophages (BMDMs) that were harvested from WT or CaMKK2 KO mice and cultured in normal media with or without 10 mM lactic acid for 96 hours. Protein from each cell type was resolved on a 7.5% acrylamide gel, and CaMKK2 or ȕ-actin was detected via Western immunoblotting. FIGS.15D-15E graphically show flow cytometry results for splenic NK cells from WT BL / 6 females that were cultured in normal media (NM), 50% E0771 TCM (TCM), or normal media with 10 mM lactic acid (LA), along with 2.5 μM CC3240 or DMSO vehicle. FIG.15D graphically shows NK cell viability. FIG.15E graphically shows expression of Ki67 at 96 hours. N=3, standard error shown.Docket No.028193-0031-WO01 / 7879

[0028] FIGS.16A-16E show that NK cells express pH sensors and lactate import channels. FIGS.16A-16E graphically show mRNA sequencing data from the Immunological Genome Project (ImmGen) that was used to interrogate expression of the GPR81 lactate sensor (FIG.16A), the GPR65 and GPR132 pH sensors (FIGS.16B-16C), and the MCT1 and MCT4 lactate transporters in various immune cells (FIGS.16D-16E). Sp= spleen, Bl= Blood, BM= bone marrow, Mo= monocyte, MF= macrophages.

[0029] FIGS.17A-17D show that intracellular lactate import is necessary for CaMKK2 upregulation. FIGS.17A-17B graphically show flow cytometry analysis data for splenic NK cells from CaMKK2-eGFP reporter mice that were cultured in normal media, normal media containing 10 mM lactic acid, or normal media containing 10 mM sodium lactate (Na lactate). Cells were also treated with 500 nM AZD3965 (AZD) or DMSO. Flow cytometry was used to measure eGFP expression relative to the NM group at 24 hours in culture (FIG.17A) and at 120 hours in culture (FIG.17B). The raw fluorescence peaks at 120 hours are shown. N=3, standard error shown. FIG.17C shows Western immunoblot analysis for bone marrow- derived macrophages (BMDMs) were harvested from WT and CaMKK2 KO mice and differentiated in normal media with M-CSF. Splenic NK cells were harvested from WT BL / 6 females and cultured in heavily buffered normal media with a fixed pH of 6.4 or 7.4 (“NM” lanes) or in heavily buffered normal media with a fixed pH of 7.4 containing 10 mM lactic acid (“Lactic acid” lanes). Various samples of lactic acid-cultured NK cells were simultaneously cultured with 2.5 μM CC3240, 500 nM AZD3965 (AZD), 5 mM DCHC (DCHC), or DMSO vehicle. CaMKK2 and the ȕ-actin control were measured via Western immunoblotting on a 7.5% acrylamide gel (45 ug protein per lane). FIG.17D graphically shows the flow cytometry results for splenic NK cells that were harvested from WT and CaMKK2 KO BL / 6 females and cultured for 96 hours in 50% EO771 TCM, heavily buffered normal media with a pH of 6.0, or heavily buffered normal media with a pH of 7.0. The total number of live NK cells, normalized to the WT level, was measured via flow cytometry. N=2, standard error shown.

[0030] FIGS.18A-18E show that the role of CaMKK2 in human NK cells is unclear. FIG. 18A shows a Western immunoblot (7.5% acrylamide gel) for measuring NK92 cells were cultured in normal growth media or 50% MDA-231 TCM for 96 hours, and CaMKK2 and ȕ- actin levels. WT MDA231 and CaMKK2 KO MDA231 cells were run on the same Western. FIGS.18B-18C show flow cytometry and Western immunoblot analyses for NK92 cells were cultured in 50% MDA-231 TCM for 96 hours with CC3240 or a DMSO vehicle. FIG.18B graphically shows that flow cytometry was used to determine viability, Ki67 expression, and Granzyme B levels. N=2, standard error shown. FIG.18C shows a Western immunoblotDocket No.028193-0031-WO01 / 7879 (7.5% acrylamide gel) for measuring CaMKK2 and ȕ-actin levels. FIGS.18D-18E show Uniform Manifold Approximation and Projection (UMAP) dimension results for immune cells from three primary human breast tumors that were subjected to single cell RNA sequencing, and the resulting data was concatenated and plotted using the UMAP dimension reduction technique. FIG.18D highlights cells expressing Ncr1 (NKp46) mRNA. FIG.18E highlights cells expressing CaMKK2 mRNA.

[0031] FIGS.19A-19B show that tumor-bearing mice engrafted with CaMKK2-deficient NK cells have fewer splenic NK cells. FIG.19A graphically shows flow cytometry analysis data where NSG mice were intravenously administered B16-F10 tumor cells and NK cells with tamoxifen-induced CaMKK2 ablation (ERT2+) or a WT control (ERT2í). After 17 days, spleens were recovered from these mice, and the total number and viability of splenic NK cells was measured using flow cytometry. N=6, standard error shown. FIG.19B graphically shows flow cytometry analysis data where NSG mice were intravenously administered B16- F10 tumor cells and WT or CaMKK2 KO NK cells. After 15 days, spleens were recovered from these mice, and the total number and viability of splenic NK cells was measured using flow cytometry. N=7-8, standard error shown. DETAILED DESCRIPTION

[0032] Tumor cells often express CaMKK2 ectopically, commandeering this enzyme to promote survival, proliferation, and metastasis across a wide variety of cancers. CaMKK2 has additional roles in tumor immunity. For example, expression of CaMKK2 promotes M2 polarization of tumor-associated macrophages and facilitates the development of myeloid- derived suppressor cells, indirectly promoting tumor growth. Pharmacologic inhibition of CaMKK2 can prevent the accumulation of M2-like tumor-associated macrophages while decreasing the number of myeloid-derived suppressor cells in the tumor microenvironment. CaMKK2 can contribute to tumor biology through its ability to regulate cancer cell-intrinsic biology and through its actions in speci¿c myeloid populations. Thus, there has been considerable interest in developing CaMKK2 inhibitors, including competitive kinase antagonists and ligand-directed degraders (LDDs), as potential cancer therapeutics.

[0033] In contrast, the inventors have discovered that natural killer (NK) cells upregulate CaMKK2 expression in the tumor environment, conferring a fitness advantage that improves anti-tumor immune activity. As detailed herein, the inventors have shown that CaMKK2 is expressed at low levels in NK cells isolated from murine spleens, but its expression is dramatically increased in NK cells isolated from tumors or exposed to tumor conditions. Further, CaMKK2 expression suppresses apoptosis and promotes proliferation of NK cellsDocket No.028193-0031-WO01 / 7879 without affecting the cytotoxic efficacy of individual cells. NK cell-intrinsic deletion of CaMKK2 increases metastatic progression across several murine models, establishing a role for CaMKK2 in NK cell tumor immunity. Ablation of the CaMKK2 protein, but not inhibition of its kinase activity, resulted in decreased NK cell survival, revealing a unique scaffold function for this enzyme. Lactic acid was identified as a driver of CaMKK2 upregulation under tumor conditions. Intracellular importation of lactic acid through the MCT channels, and not general acidification, may play a role in CaMKK2 upregulation in NK cells. These results suggested that CaMKK2 upregulation in tumor-infiltrating NK cells may be an adaptive mechanism by which these cells mitigate the deleterious effects of a lactate-rich tumor environment.

[0034] Described herein are compositions and methods to increase the viability of natural killer (NK) cells. The compositions and methods may increase the growth, increase the replication rate, increase the proliferation, or decrease cell death or apoptosis in the NK cell. The compositions and methods may be used to treat cancer in a subject. 1. Definitions

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0036] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0037] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the rangeDocket No.028193-0031-WO01 / 7879 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0038] The term “about” or “approximately” as used herein as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In certain aspects, the term “about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Alternatively, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value.

[0039] “Adeno-associated virus” or “AAV” as used interchangeably herein refers to a small virus belonging to the genus Dependovirus of the Parvoviridae family that infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response.

[0040] The term “administration” or “administering,” as used herein refers to providing, contacting, and / or delivery an agent or composition as detailed herein, by any appropriate route to achieve the desired effect. These agents may be administered to a subject in numerous ways and may be used in combination.

[0041] “Amino acid” as used herein refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids can be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acids include the side chain and polypeptide backbone portions.

[0042] The terms “cancer”, “cancer cell”, “tumor”, and “tumor cell” are used interchangeably herein and refer generally to a group of diseases characterized by uncontrolled, abnormal growth of cells (for example, a neoplasia). In some forms of cancer,Docket No.028193-0031-WO01 / 7879 the cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body (“metastatic cancer”). “Cancer” refers to all types of cancer or neoplasm or malignant tumors found in animals, including carcinoma, adenoma, melanoma, sarcoma, lymphoma, leukemia, blastoma, glioma, astrocytoma, mesothelioma, or a germ cell tumor. Cancer may include cancer of, for example, the colon, rectum, stomach, bladder, cervix, uterus, skin, epithelium, muscle, kidney, liver, lymph, bone, blood, ovary, prostate, lung, brain, head and neck, and / or breast. Cancer may include medullablastoma, non-small cell lung cancer, and / or mesothelioma. In embodiments detailed herein, the cancer includes leukemia. The term “leukemia” refers to broadly progressive, malignant diseases of the hematopoietic organs / systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia diseases include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, and promyelocytic leukemia. In some embodiments, the leukemia is chronic myeloid leukemia (CML). In some embodiments, the leukemia is acute myeloid leukemia (AML).

[0043] “Coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The regulatory elements may include, for example, a promoter, an enhancer, an initiation codon, a stop codon, or a polyadenylation signal. The coding sequence may be codon optimized.Docket No.028193-0031-WO01 / 7879

[0044] “Complement” or “complementary” as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0045] The terms “control,” “reference level,” and “reference” are used herein interchangeably. The reference level may be a predetermined value or range, which is employed as a benchmark against which to assess the measured result. “Control group” as used herein refers to a group of control subjects. The predetermined level may be a cutoff value from a control group. The predetermined level may be an average from a control group. Cutoff values (or predetermined cutoff values) may be determined by Adaptive Index Model (AIM) methodology. Cutoff values (or predetermined cutoff values) may be determined by a receiver operating curve (ROC) analysis from biological samples of the patient group. ROC analysis, as generally known in the biological arts, is a determination of the ability of a test to discriminate one condition from another, e.g., to determine the performance of each marker in identifying a patient having CRC. A description of ROC analysis is provided in P.J. Heagerty et al. (Biometrics 2000, 56, 337-44), the disclosure of which is hereby incorporated by reference in its entirety. Alternatively, cutoff values may be determined by a quartile analysis of biological samples of a patient group. For example, a cutoff value may be determined by selecting a value that corresponds to any value in the 25th-75th percentile range, preferably a value that corresponds to the 25th percentile, the 50th percentile or the 75th percentile, and more preferably the 75th percentile. Such statistical analyses may be performed using any method known in the art and can be implemented through any number of commercially available software packages (e.g., from Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station, TX; SAS Institute Inc., Cary, NC.). The healthy or normal levels or ranges for a target or for a protein activity may be defined in accordance with standard practice. A control may be a subject or cell without a composition as detailed herein. A control may be a subject, or a sample therefrom, whose disease state is known. The subject, or sample therefrom, may be healthy, diseased, diseased prior to treatment, diseased during treatment, or diseased after treatment, or a combination thereof.

[0046] The term “disease” as used herein includes, but is not limited to, any abnormal condition and / or disorder of a structure or a function that affects a part of an organism. It may be caused by an external factor, such as an infectious disease, or by internal dysfunctions, such as cancer, cancer metastasis, and the like.Docket No.028193-0031-WO01 / 7879

[0047] “Effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0048] “Genetic construct" as used herein refers to the DNA or RNA molecules that comprise a polynucleotide that encodes a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed. The regulatory elements may include, for example, a promoter, an enhancer, an initiation codon, a stop codon, or a polyadenylation signal.

[0049] “Identical” or “identity” as a percentage as used herein in the context of two or more polynucleotide or polypeptide sequences means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0050] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a polynucleotide also encompasses the complementary strand of a depicted single strand. Many variants of a polynucleotide may be used for the same purpose as a given polynucleotide. Thus, a polynucleotide also encompasses substantially identical polynucleotides and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions. Polynucleotides may be single stranded or double stranded or may contain portions of both double stranded and single strandedDocket No.028193-0031-WO01 / 7879 sequence. The polynucleotide can be nucleic acid, natural or synthetic, DNA, genomic DNA, cDNA, RNA, mRNA, or a hybrid, where the polynucleotide can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including, for example, uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.

[0051] “Open reading frame” refers to a stretch of codons that begins with a start codon and ends at a stop codon. In eukaryotic genes with multiple exons, introns are removed, and exons are then joined together after transcription to yield the final mRNA for protein translation. An open reading frame may be a continuous stretch of codons. In some embodiments, the open reading frame only applies to spliced mRNAs, not genomic DNA, for expression of a protein.

[0052] “Operably linked” as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function. Nucleic acid or amino acid sequences are “operably linked” (or “operatively linked”) when placed into a functional relationship with one another. For instance, a promoter or enhancer is operably linked to a coding sequence if it regulates, or contributes to the modulation of, the transcription of the coding sequence. Operably linked DNA sequences are typically contiguous, and operably linked amino acid sequences are typically contiguous and in the same reading frame. However, since enhancers generally function when separated from the promoter by up to several kilobases or more and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous. Similarly, certain amino acid sequences that are non-contiguous in a primary polypeptide sequence may nonetheless be operably linked due to, for example folding of a polypeptide chain. With respect to fusion polypeptides, the terms “operatively linked” and “operably linked” can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked.

[0053] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide”, “protein,” and “peptide”Docket No.028193-0031-WO01 / 7879 are used interchangeably herein. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, for example, enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. “Domains” are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha- helices. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units. A “motif” is a portion of a polypeptide sequence and includes at least two amino acids. A motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids in length. In some embodiments, a motif includes 3, 4, 5, 6, or 7 sequential amino acids. A domain may be comprised of a series of the same type of motif.

[0054] “Promoter” as used herein means a synthetic or naturally derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, human U6 (hU6) promoter, and CMV IE promoter. Promoters that target muscle-specific stem cells may include the CK8 promoter, the Spc5-12 promoter, and the MHCK7 promoter.

[0055] The term “recombinant” when used with reference to, for example, a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, forDocket No.028193-0031-WO01 / 7879 example, recombinant cells express genes that are not found within the native (naturally occurring) form of the cell or express a second copy of a native gene that is otherwise normally or abnormally expressed, under expressed, or not expressed at all.

[0056] “Sample” or “test sample” as used herein can mean any sample in which the presence and / or level of a target is to be detected or determined or any sample comprising a DNA targeting or gene editing system or component thereof as detailed herein. Samples may include liquids, solutions, emulsions, or suspensions. Samples may include a medical sample. Samples may include any biological fluid or tissue, such as blood, whole blood, fractions of blood such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid, skin, or combinations thereof. In some embodiments, the sample comprises an aliquot. In other embodiments, the sample comprises a biological fluid. Samples can be obtained by any means known in the art. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

[0057] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal that wants or is in need of the herein described compositions or methods. The subject may be a human or a non-human. The subject may be a vertebrate. The subject may be a mammal. The mammal may be a primate or a non- primate. The mammal can be a non-primate such as, for example, cow, pig, camel, llama, hedgehog, anteater, platypus, elephant, alpaca, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, and mouse. The mammal can be a primate such as a human. The mammal can be a non-human primate such as, for example, monkey, cynomolgous monkey, rhesus monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, a child, such as age 0-2, 2-4, 2-6, or 6-12 years, or an infant, such as age 0-1 years. The subject may be male. The subject may be female. In some embodiments, the subject has a specific genetic marker. The subject may be undergoing other forms of treatment. In some embodiments, the subject has cancer.

[0058] “Substantially identical” can mean that a first and second amino acid or polynucleotide sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,Docket No.028193-0031-WO01 / 7879 97%, 98%, or 99%, or less than 100% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 amino acids or nucleotides, respectively.

[0059] “Transcriptional regulatory elements” or “regulatory elements” refers to a genetic element which can control the expression of nucleic acid sequences, such as activate, enhancer, or decrease expression, or alter the spatial and / or temporal expression of a nucleic acid sequence. Examples of regulatory elements include, for example, promoters, enhancers, splicing signals, polyadenylation signals, and termination signals. A regulatory element can be “endogenous,” “exogenous,” or “heterologous” with respect to the gene to which it is operably linked. An “endogenous” regulatory element is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” regulatory element is one which is not normally linked with a given gene but is placed in operable linkage with a gene by genetic manipulation.

[0060] “Treatment” or “treating” or “therapy” when referring to protection of a subject from a disease, means suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Treatment may result in a reduction in the incidence, frequency, severity, and / or duration of symptoms of the disease. Preventing the disease involves administering a composition of the present invention to a subject prior to onset of the disease. Suppressing the disease involves administering a composition of the present invention to a subject after induction of the disease but before its clinical appearance. Repressing or ameliorating the disease involves administering a composition of the present invention to a subject after clinical appearance of the disease.

[0061] “Variant” used herein with respect to a polynucleotide means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequence substantially identical thereto. A variant can be a polynucleotide sequence that is substantially identical over the full length of the full polynucleotide sequence or a fragment thereof. The polynucleotide sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,Docket No.028193-0031-WO01 / 7879 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or less than 100% identical over the full length of the polynucleotide sequence or a fragment thereof.

[0062] “Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific antibody or polypeptide or to promote an immune response. Variant can mean a functional fragment thereof. Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem or separated by a linker. A conservative substitution of an amino acid, for example, replacing an amino acid with a different amino acid of similar properties (for example, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes may be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (Kyte et al., J. Mol. Biol.1982, 157, 105-132). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes may be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids may also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or less than 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0063] “Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector may be capable of directing the delivery or transfer of a polynucleotide sequence to target cells, where it can be replicated or expressed. A vector may contain anDocket No.028193-0031-WO01 / 7879 origin of replication, one or more regulatory elements, and / or one or more coding sequences. A vector may be a viral vector, bacteriophage, bacterial artificial chromosome, plasmid, cosmid, or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector. Viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus (AAV) vector, retrovirus vector, or lentivirus vector. A vector may be an adeno-associated virus (AAV) vector.

[0064] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. 2. Natural Killer (NK) Cells

[0065] Natural killer (NK) cells perform a range of unique anti-pathogen and anti-tumor functions in the immune system. Like T-cells and B-cells, NK cells arise from common lymphoid progenitors in the bone marrow before trafficking to the lymphoid organs, yet NK cells adopt a distinct granular morphology and function as part of the innate immune system. NK cells are not antigen-specific, do not undergo diversification and selection processes, and do not require activation signals from antigen-presenting cells. Rather, NK cells directly integrate activation and suppression signals through germline-encoded cell-surface receptors that are paired with cytoplasmic ITIM and ITAM motifs to trigger effector activity. Upon proper stimulation, NK cells mediate cytotoxic killing in the same manner as CD8 T- cells. NK cells release pre-formed granules containing perforin, which forms channels in the membrane of the target cells, and granzyme, which enters those channels and directly cleaves caspase 3, triggering apoptosis. NK cells also produce FasL and TRAIL, which trigger the extrinsic apoptosis pathway if the cognate receptors remain intact on the target cell. The primary function of NK cells is to eliminate cells that have lost or downregulated MHC class I and are thus missing “self” markers. For example, cells engineered to lose MHC class I expression are readily cleared in mice, which is a function dependent on the natural killer population. Loss of normal MHC class I expression is a marker of pathologicalDocket No.028193-0031-WO01 / 7879 transformation, as this complex is the primary means by which CD8 T-cells surveil cytoplasmic peptide content.

[0066] NK cell activity may be regulated by a variety of receptors on the NK cell surface. Receptors on NK cells that activate the NK cell to attack a target cell are referred to as activating receptors, which may include members of the natural cytotoxicity receptor (NCR) family, such as NKG2D, NKp46, and NKp30, and combinations thereof. Receptors on NK cells that inhibit the NK cell from attacking a target cell are referred to as inhibitory receptors, which may include members of the killer immunoglobulin-like receptor (KIR) family. KIR receptors recognize a variety of MHC class I alleles (HLA in humans) that is expressed on all healthy nucleated cells. As detailed above, expression of the proper MHC class I allele labels a cell as “self” and strongly inhibits NK cell cytotoxicity.

[0067] NK cells may arise from common lymphoid progenitors (CLPs) in the bone marrow and differentiate into a variety of subpopulations with distinct functions. CLPs become committed to NK differentiation upon expression of CD122 (IL-2Rȕ), and subsequent development in the bone marrow is largely driven by IL15 signaling. In the bone marrow, NK cells undergo the “licensing” or “education” process by which they gain cytotoxic effector capabilities contingent on expression of inhibitory KIR receptors, preventing autoimmune sequelae. After leaving the bone marrow, peripheral NK cells continue to progress through additional stages of maturation in which effector capacity in augmented. In mice, “immature” NK cells are CD11b- / CD27+, whereas mature type 1 cells are CD11b+ / CD27+, and the highly cytolytic mature type 2 cells are CD11b+ / CD27-. Human NK cells may also express markers including CD11b, CD27, CD16 (FcȖRIII), and the human- specific marker CD56. Human NK cells gain increasing cytolytic function as they progress from CD56brightCD16- to CD56dimCD16dimto CD56dimCD16bright 30. To acquire specific functions, subsets of NK cells will pause at particular maturation stages or embark on entirely divergent pathway. For example, CD56brightCD16dimNK cells are prolific producers of cytokines and chemokines, activating and recruiting a variety of immune cells. NK cells may serve unique tissue-specific roles, such as their role in placenta vascularization during pregnancy. These “decidual” NK cells reside at the placenta-uterine interface and produce copious amounts of pro-angiogenic factors such as those from the VEGF family. Natural killer T (NKT) cells are most often classified as a subpopulation of T cells but may also be classified as an NK subset. NKT cells may undergo thymic development and express a TCR like T-cells, yet these cells are innately cytotoxic, express most canonical NK receptors, and their classic TCR is an invariant ligand of CD1d that essentially functions as an NK activation receptor.Docket No.028193-0031-WO01 / 7879

[0068] Tumor cells may adopt a similar strategy to hide their mutated neo-antigens from T-cell surveillance, by reducing MHC Class I expression through genomic and epigenomic modifications to various steps in the MHC Class I antigen presentation pathway. NK cells may thereby serve as a complement to T-cell immunity, killing tumors that evade T cells through MHC Class I downregulation. However, NK cells may also require engagement of activation receptors to mediate cytotoxic release. Activating ligands may include an assortment of DNA damage markers, such as, for example, MICA, MICB, and ULBP1-3, which may be expressed on tumor cells.

[0069] NK cells may have additional functions. For example, NK cells and monocytes are the mediators of antibody-dependent cellular cytotoxicity (ADCC), the process by which antibody-opsonized cells are lysed. NK cells may detect the constant domain (Fc) of cell- bound IgG antibodies via their Fc receptors (CD16), producing a powerful activation signal that leads to degranulation. Certain subsets of NK cells may be specialized for cytokine signaling rather than direct cytotoxicity, for example, the CD56brightCD16- subset of human NK cells. These cells may produce IFNȖ, which increases MHC Class I expression across cell types while promoting activation and differentiation of various immune cells. NK cells may also produce Flt3L, which may be a chemoattractant for dendritic cells. Another subset of NK cells, termed “regulatory” NK cells and defined by CD73 expression, may promote tumor growth by producing immune suppressive cytokines such as IL10 and expressing checkpoint markers like LAG-3.

[0070] The metabolic state and effector function of NK cells may be regulated by, for example, the competing effects of the AKT / mTOR and AMPK pathways. The AKT / mTORc1 pathway may promote an activated glycolytic state characterized by enhanced glucose uptake, increased oxidative phosphorylation, elevated production of IFNȖ and Granzyme B, and augmented effector activity against tumor cells. Together with the IL15 / JAK / STAT5 pathway, the AKT / mTORc1 pathway may regulate maturation of NK cells into later effector stages, and mTOR may regulate the “education” process that maintains NK cell reactivity through frequent MHC I engagment. A variety of activation receptors, including NKG2D, CD16, IL15, and IL10, may activate the mTORc1 pathway by triggering PI3K and AKT signaling. AKT may regulate some processes independently of mTOR, such as promoting cell-cycle progression through regulation of cyclin D3 transcription, E2F activation, and cytoplasmic sequestration of p27.

[0071] The AMPK pathway may promote an inactive, quiescent state in response to energy deprivation or suppressive signals. AMPK activation in NK cells is characterized by suppressed IFNȖ production, decreased activation marker expression, reduced proliferation,Docket No.028193-0031-WO01 / 7879 and diminished ex vivo cytotoxic activity. AMPK may be activated by an elevated AMP / ATP ratio, attenuating energy-demanding cellular functions when energy is in short supply, but in NK cells, AMPK may be also activated by adiponectin signaling and engagement of certain inhibitory receptors such as KLGR1. The suppressive effects of AMPK may be mediated through inhibition of NF-^B signaling and through antagonism of the mTOR pathway. The TSC1 / TSC2 complex may be a major negative regulator of mTOR that integrates competing signals from the AKT1 and AMPK pathways. AMPK phosphorylates TSC2 at Thr1227 and Ser1345, driving TSC2 activation and mTOR inhibition, whereas AKT1 phosphorylates TSC2 at Ser939 and Thr1462, driving TSC2 inhibition and mTOR activation. The AKT / mTOR and AMPK pathways are competing regulators of NK cell function.

[0072] Allogeneic NK cell transfusions may be used according to the compositions and methods detailed herein. NK cells can be fitted with chimeric antigen receptors (CARs) in the same or similar manner as T-cells to mediate cytotoxicity against specific antigen targets, referred to as CAR-NK therapies. Because CAR-NK cells lack a TCR and have a limited lifespan in circulation, they may carry a reduced risk of graft-versus-host disease, cytokine release syndrome, and neurotoxicity compared to CAR-T cells (Xie, G. et al. EBioMedicine 2020, 59, 102975, incorporated herein by reference). CAR-NK cells can be engineered by a variety of methods known in the art, for example, from primary NK cells or from cell lines such as NK92, which is a constitutively active human NK cell lymphoma line with attenuated expression of the KIR inhibitory receptors (Wu, L., et al. Int. Immunopharmacol.2011, 11, 274-227, incorporated herein by reference). NK92 cells may be used in ways other than CAR modification (Suck, G. et al. Cancer Immunol. Immunother. 2016, 65, 485-492; Hoerster, K. et al. Front. Immunol.2020, 11, 586168; Sayitoglu, E. C. et al. Front. Immunol.2020, 11, 40; each incorporated herein by reference). NK cells and methods of isolating, modifying, and / or administering to a subject are described in, for example, PCT Application Publication Nos. WO2019 / 213610, WO2009 / 126789, WO2012 / 009422, and WO2013 / 094988, each of which is incorporated herein by reference.

[0073] Provided herein are modified NK cells. The NK cells may be modified to have increased survival and / or proliferation relative to a non-modified control. The modified NK cells may have improved survival and / or activity, for example, in the conditions created by solid tumors, which may have a hypoxic, nutrient-depleted, immune suppressive tumor microenvironment. The NK cells may be autologous, for example, extracted and / or derived from a subject and them modified for use in the same subject. The NK cells may be heterologous, such as including or being derived from a cell line such a NK92 cells. The NK cells may be modified to have increased expression or activity of Calcium / calmodulin-Docket No.028193-0031-WO01 / 7879 dependent protein kinase kinase 2 (CaMKK2). The NK cells may include additional modifications. For example, the NK cells may be engineered to have reduced or eliminated expression of the TGF-ȕ receptor, which is a suppressor of NK cell activity within solid tumors (Viel, S. et al. Sci. Signal 2016, 9, ra19; Yang, B. et al. Int. Immunopharmacol.2013, 17, 198-204; each incorporated herein by reference), and reduced or eliminated expression of the TGF-ȕ receptor may enhance NK cell anti-tumor activity and attenuate tumor growth. As another example, the NK cells may be engineered to have increased expression of LDH.

[0074] NK cells may be isolated from a subject by any suitable means known in the art. A sample may be taken from the subject. The sample may comprise blood, plasma, tissue, tissue from a tumor, tumor biopsy, or a combination thereof. In some embodiments, the sample comprises blood. The blood may be peripheral blood. The blood may comprise peripheral blood mononuclear cells (PBMCs). The sample may comprise plasma. The sample may comprise serum. NK cells may be isolated from the sample by any suitable means known in the art. For example, PBMCs may be separated from whole patient blood through leukapheresis. Anti-CD3 immunomagnetic particles may be used to deplete T-cells, and the remaining PBMCs may be co-cultured for 2 weeks with irradiated IL-15 expressing K562 to expand the NK cell fraction. Once modified, and as further detailed below, modified NK cells may be administered to a subject by any suitable means known in the art, for example, modified NK cells may be injected or autologously transplanted back into the original patient or donor. In some embodiments, isolated NK cells are grown in cell culture, maintained in cell media, and / or expanded ex vivo. NK cells may be identified by positive or negative expression of cell surface markers. A NK cell may be identified by being, for example, CD56+, CD3-, or a combination thereof. Cell surface markers may be identified by any suitable means known in the art, such as, for example, flow activated cell sorting (FACS), flow cytometry, immunohistochemistry, and binding to labelled antibodies. a. CaMKK2

[0075] The NK cells, for example, isolated NK cells from a subject, may be modified to have increased expression or activity of calcium / calmodulin-dependent protein kinase kinase 2 (CaMKK2). CaMKK2 is a calcium-activated serine / threonine kinase and calcium-activated regulator of energy homeostasis in many cell types, including neurons and hepatocytes. CaMKK2 is activated by a cytoplasmic calcium influx that triggers calmodulin binding. Calmodulin binding triggers conformational changes that expose target-binding domains, allowing CaMKK2 to phosphorylate downstream targets such as CaMKI, CaMKIV, AKT, and AMPK.Docket No.028193-0031-WO01 / 7879

[0076] CaMKK2 expression is most prevalent in the central nervous system (CNS) where it activates the CaMKI and CaMKIV pathways to regulate neuronal development and survival. CaMKK2 may promote neuron elongation and branching through CaMKI signaling, with different CaMKK2 isoforms favoring distinct neuron morphologies. CaMKK2 may be expressed in several tissues outside the CNS where it may regulate both cellular and whole- body metabolism, largely via the AMPK pathway. For example, CaMKK2 expression in the hypothalamus has a role in the production of hormone neuropeptide Y, a potent appetite stimulant that specifically favors carbohydrate intake. In pancreatic ȕ-islet cells, CaMKK2 regulates insulin release by sensing calcium fluctuations, and CaMKK2 promotes gluconeogenesis in hepatocytes, likely by coupling catecholamine signaling to the AMPK pathway. CaMKK2 also plays a role in skeletal homeostasis, enabling osteoblast activation via sympathetic neuronal signaling while directly inhibiting osteoclast differentiation. CaMKK2 deletion may protect against ovariectomy-induced osteoporosis in mice. CaMKK2 signaling may attenuate the differentiation of adipose and muscle tissue, preventing myoblast and pre-adipocytes from initiating differentiation programs.

[0077] Tumor cells tend to express high levels of CaMKK2, which confers a variety of selective advantages to promote tumor growth, proliferation, and / or metastasis. In many tumors, CaMKK2 bypasses PDK1 and directly activates the AKT pathway, promoting tumor cell survival in prostate tumors while enabling carboplatin resistance and apoptosis suppression in ovarian cancers. In LKB1-deficient lung cancer, CaMKK2 is upregulated by the PLAG1 transcription factor to suppress anoikis and enable metastatic dissemination. In breast cancer, CaMKK2 expression may promote tumor cell migration. In prostate cancer, CaMKK2 has a role in androgen-mediated tumor cell migration. CaMKK2 may regulate golgi trafficking in prostate tumors, preventing ER dysfunction and preserving proliferative capacity. In hepatocellular carcinoma (HCC), CaMKK2 may promote proliferation by providing a scaffold upon which CaMKIV, S6, S6K, and mTOR can assemble and regulate the cell cycle. CaMKK2 expression in melanoma cells may reduce lipid peroxidation and ferroptotic cell death through NRF2 / AMPK activation. CaMKK2 expression may also favor M2 polarization of tumor-associated macrophages (TAMs). Treatment with the CaMKK2 inhibitor STO609 reduces primary breast tumor growth in mice while increasing the prevalence of inflammatory M1 macrophages.

[0078] Various CaMKK2 inhibitors are known in the art and may be obtained commercially. CaMKK2 inhibitors may include competitive inhibitors such as competitors of ATP binding that inhibit the kinase activity of CaMKK2. For example, the CaMKK2 antagonist STO609 is a competitive inhibitor of ATP binding. However, STO609 has aDocket No.028193-0031-WO01 / 7879 relatively low binding affinity for CaMKK2 and mediates several off-target effects, including direct inhibition of AMPK. Selective, high-affinity kinase inhibitors include, for example, GSK1901320, GSK650934, 4t, YL-36, SOB8-17-3, and CC8977. Another class of CaMKK2 inhibitors are ligand-directed degraders (LDDs). LDDs are bi-specific molecules that feature a target-binding domain paired with a ubiquitin ligase recruitment domain. By bringing the E3 ubiquitin ligase in proximity to CaMKK2, the kinase may be polyubiquitylated and degraded through the proteasomal pathway, thus eliminating the entire protein rather than selectively inhibiting the kinase domain (Martin-Acosta, P. et al. Eur. J. Med. Chem.2021, 210, 112993, incorporated by reference). Compared to classic kinase inhibitors, LDDs may offer advantages such as increased target selectivity and activity against inhibitor-resistant tumors. Other routes for inhibiting CaMKK2 may include fungus-derived fusicoccins, which stabilize interactions between CaMKK2 and the inhibitory regulator 14-3-3Ȗ (Lentini Santo, D., et al. ACS Chem. Biol.2020, 15, 3060-3071, incorporated by reference).

[0079] NK cells may be modified by administering or introducing to the NK cell a polynucleotide encoding CaMKK2. CaMKK2 may comprise a polypeptide having the amino acid sequence of SEQ ID NO: 1. CaMKK2 may be encoded by a polynucleotide comprising the sequence of SEQ ID NO: 2 or 3. NK cells may be modified by mRNA transfection to transiently overexpress CaMKK2 in the NK cells. The polynucleotide encoding CaMKK2 may be comprised within a genetic construct. The genetic construct may be administered or introduced to the NK cell by any suitable means known in the art, for example, an expanded population of NK cells may be transduced with a retroviral construct promoting constitutive CaMKK2 expression. Upon administration of the polynucleotide encoding CaMKK2, CaMKK2 may be expressed in the NK cell.

[0080] A polynucleotide encoding a CaMKK2 protein can be a synthetic polynucleotide. For example, the synthetic polynucleotide can be chemically modified. The synthetic polynucleotide can be codon optimized, for example, at least one non-common codon or less-common codon has been replaced by a common codon. For example, the synthetic polynucleotide can direct the synthesis of an optimized messenger mRNA, for example, optimized for expression in a mammalian expression system, as described herein. A variety of suitable promoters may be used. For example, the genetic construct comprising the polynucleotide encoding a CaMKK2 may include a constitutive promoter directing expression of the CaMKK2 gene. In some embodiments, the modified NK cell constitutively expresses a level of CaMKK2 greater than that of a control NK cell. The genetic construct may include a tissue-specific promoter directing expression of the CaMKK2 gene to a particular cell and / or tissue.Docket No.028193-0031-WO01 / 7879

[0081] The modified NK cell may express a level of CaMKK2 greater than a control. A control may be an unmodified NK cell. The control may be an average level of expression of CaMKK2 in unmodified NK cell(s). A control may be an unmodified NK cell from a healthy subject. A control may be an unmodified NK cell from a subject with cancer. The expression of CaMKK2 may be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5- fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold greater than that of a control. The expression of CaMKK2 may be increased by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The expression of CaMK2 may be increased by about 5-95%, 10-90%, 15-85%, 20-80%, or 1.5-fold to 10-fold, relative to a control.

[0082] An activity of CaMKK2 in the modified NK cell may be increased relative to a control. A control may be an unmodified NK cell. The control may be an average level of expression of CaMKK2 in unmodified NK cell(s). A control may be an unmodified NK cell from a healthy subject. A control may be an unmodified NK cell from a subject with cancer. The activity of CaMKK2 may be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5- fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold greater than that of a control. The activity of CaMKK2 may be increased by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The activity of CaMK2 may be increased by about 5-95%, 10-90%, 15-85%, 20-80%, or 1.5-fold to 10-fold, relative to a control. In some embodiments, the activity relates to ATP hydrolysis activity and may include ATP binding, ADP release, and / or ATP hydrolysis. In some embodiments, the ATP hydrolysis activity of the CaMKK2 is not modified relative to a control. In some embodiments, an activity of CaMKK2, other than ATP hydrolysis and / or binding, in the modified NK cell may be increased relative to a control. In some embodiments, the ATP hydrolysis activity of the CaMKK2 is abolished or reduced relative to a control. In some embodiments, the activity relates to a scaffold function of CaMKK2. In some embodiments, the binding of CaMKK2 to another polypeptide or small molecule is increased relative to a control. In some embodiments, the binding of CaMKK2 to another polypeptide or small molecule is maintained relative to a control. The polypeptide may include a receptor, a cellDocket No.028193-0031-WO01 / 7879 surface receptor, or other protein. In some embodiments, CaMKK2 expressed in the modified NK cell maintains scaffolding activity. b. Lactate

[0083] Intracellular lactate import into the NK cells may be needed for CaMKK2 upregulation. Lactic acid or lactate may be administered to the NK cell to modify it to have an increased level of CaMKK2 in the NK cell. NK cells may be isolated from a subject, and cultured in cell media including an increased level of lactic acid or lactate in order to increase the concentration of lactic acid in the NK cell. The increased concentration of lactic acid or lactate in the NK cell may increase the expression of CaMKK2 in the NK cell. Healthy tissues and blood may have a lactic acid or lactate concentration of at least about 1 mM, at least about 1.5 mM, at least about 2 mM, at least about 2.5 mM, or at least about 3 mM. Healthy tissues and blood may have a lactic acid or lactate concentration of less than about 1 mM, less than about 1.5 mM, less than about 2 mM, less than about 2.5 mM, less than about 3 mM, or less than about 3.5 mM. Healthy tissues and blood may have a lactic acid or lactate concentration of about 1 mM to about 3.5 mM, or about 1.5 mM to about 3 mM. A lactic acid or lactate concentration of at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, or at least about 30 mM may be representative of a typical solid tumor in humans. A lactic acid or lactate concentration of less than about 30 mM, less than about 25 mM, less than about 20 mM, less than about 15 mM, or less than about 12 mM may be representative of a typical solid tumor in humans. The concentration of lactic acid or lactate in the NK cell medium may be at least about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM to increase the expression of CaMKK2 in the NK cell relative to a control. The concentration of lactic acid or lactate in the NK cell medium may be from about 5 mM to about 15 mM, about 8 mM to about 12 mM, about 9 mM to about 11 mM, or about 10 mM to increase the expression of CaMKK2 in the NK cell relative to a control. The concentration of lactic acid or lactate in the NK cell may be at least about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM to increase the expression of CaMKK2 in the NK cell relative to a control. The concentration of lactic acid or lactate in the NK cell may be from about 5 mM to about 15 mM, about 8 mM to about 12 mM, about 9 mM to about 11 mM, or about 10 mM to increase the expression of CaMKK2 in the NK cell relative to a control.

[0084] In some embodiments, intracellular concentration of lactate or lactic acid in a NK cell may be increased by increasing the expression of lactate dehydrogenase (LDH) in the NK cell. A NK cell may be administered a polynucleotide encoding LDH. A NK cell may be modified by administering or introducing to the NK cell a polynucleotide encoding LDH. LDHDocket No.028193-0031-WO01 / 7879 may comprise a polypeptide having the amino acid sequence of SEQ ID NO: 4. LDH may be encoded by a polynucleotide comprising the sequence of SEQ ID NO: 5 or 6. The polynucleotide encoding LDH may be comprised within a genetic construct. NK cells may be modified by mRNA transfection to transiently overexpress LDH in the NK cells. The genetic construct may be administered or introduced to the NK cell by any suitable means known in the art, for example, an expanded population of NK cells may be transduced with a retroviral construct promoting constitutive LDH expression. Upon administration of the polynucleotide encoding LDH, LDH may be expressed in the NK cell, and the intracellular concentration of lactate or lactic acid in a NK cell may be increased. The level of CaMKK2 in the NK cell may thereby be increased. The expression of LDH may be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold greater than that of a control. The expression of LDH may be increased by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The expression of LDH may be increased by about 5-95%, 10-90%, 15-85%, 20-80%, or 1.5-fold to 10-fold, relative to a control. As indicated above, controls may include an unmodified NK cell. c. Increasing Viability of NK Cells

[0085] The modified NK cell having an increased expression or activity of CaMKK2 may display a variety of properties relating to enhanced viability. As stated above, the modified NK cells may have improved survival and / or activity, for example, in the conditions created by solid tumors, which have a hypoxic, nutrient-depleted, immune suppressive tumor microenvironment. The enhanced viability of the modified NK cell may be relative to control, such as, for example, an unmodified NK cell, an average level of an activity or property in unmodified NK cell(s), an unmodified NK cell from a healthy subject, or an unmodified NK cell from a subject with cancer.

[0086] The modified NK cell may have increased viability compared to a control NK cell. The viability of the modified NK cell may be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5- fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold greater than that of a control. The viability of the modified NK cell may be increased by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-Docket No.028193-0031-WO01 / 7879 fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The viability of the modified NK cell may be increased by about 5-95%, 10-90%, 15-85%, 20- 80%, or 1.5-fold to 10-fold, relative to a control.

[0087] The modified NK cell may have a reduced rate of apoptosis compared to a control NK cell. The rate of apoptosis of the modified NK cell may be reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The rate of apoptosis of the modified NK cell may be reduced by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The rate of apoptosis of the modified NK cell may be reduced by about 5-95%, 10-90%, 15-85%, 20-80%, or 1.5-fold to 10-fold, relative to a control.

[0088] The modified NK cell may have an increased rate of replication and / or growth and / or proliferation compared to a control NK cell. The rate of replication and / or growth and / or proliferation of the modified NK cell may be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold greater than that of a control. The rate of replication and / or growth and / or proliferation of the modified NK cell may be increased by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, or 10-fold, relative to a control. The rate of replication and / or growth and / or proliferation of the modified NK cell may be increased by about 5-95%, 10-90%, 15- 85%, 20-80%, or 1.5-fold to 10-fold, relative to a control. 3. Genetic Constructs

[0089] The CaMKK2 protein may be encoded by one or more genetic constructs. The genetic construct, such as a plasmid or expression vector, may comprise a nucleic acid that encodes the CaMKK2 protein. Genetic constructs may include polynucleotides such as vectors and plasmids. The genetic construct may be a linear minichromosome including centromere, telomeres, or plasmids or cosmids. The vector may be an expression vector or system to produce protein by routine techniques and readily available starting materials including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is incorporated fully by reference. The construct may beDocket No.028193-0031-WO01 / 7879 recombinant. The genetic construct may be part of a genome of a recombinant viral vector, including recombinant lentivirus, recombinant adenovirus, and recombinant adenovirus associated virus. The genetic construct may comprise regulatory elements for gene expression of the coding sequences of the nucleic acid. The regulatory elements may be a promoter, an enhancer, an initiation codon, a stop codon, or a polyadenylation signal.

[0090] The genetic construct may comprise a heterologous nucleic acid encoding the CaMKK2 protein and may further comprise an initiation codon, which may be upstream of the CaMKK2 protein coding sequence, and a stop codon, which may be downstream of the CaMKK2 protein coding sequence. The genetic construct may include more than one stop codon, which may be downstream of the CaMKK2 protein coding sequence. A stop codon may be in-frame with a coding sequence. The initiation and termination codon may be in frame with the CaMKK2 protein coding sequence.

[0091] The vector may also comprise a promoter that is operably linked to the CaMKK2 protein coding sequence. The promoter may be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulatable promoter. The promoter may be a ubiquitous promoter. The promoter may be a tissue-specific promoter. The tissue specific promoter may be a muscle specific promoter. The tissue specific promoter may be a skin specific promoter. The CaMKK2 protein may be under the light-inducible or chemically inducible control to enable the dynamic control of expression in space and time. The promoter operably linked to the CaMKK2 protein coding sequence may be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. Examples of a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic, are described in U.S. Patent Application Publication No. US20040175727, the contents of which are incorporated herein in its entirety. The promoter may be a CK8 promoter, a Spc512 promoter, a MHCK7 promoter, for example.

[0092] The genetic construct may also comprise a polyadenylation signal, which may be downstream of the CaMKK2 protein coding sequence. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human ȕ-Docket No.028193-0031-WO01 / 7879 globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA).

[0093] Coding sequences in the genetic construct may be optimized for stability and high levels of expression. In some instances, codons are selected to reduce secondary structure formation of the RNA such as that formed due to intramolecular bonding.

[0094] The genetic construct may also comprise an enhancer upstream of the CaMKK2 protein coding sequence. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, HA, RSV, or EBV. Polynucleotide function enhancers are described in U.S. Patent Nos.5,593,972, 5,962,428, and WO94 / 016737, the contents of each are fully incorporated by reference. The genetic construct may also comprise a mammalian origin of replication in order to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell. The genetic construct may also comprise a regulatory sequence, which may be well suited for gene expression in a mammalian or human cell into which the vector is administered. The genetic construct may also comprise a reporter gene, such as green fluorescent protein (“GFP”) and / or a selectable marker, such as hygromycin (“Hygro”).

[0095] The genetic construct may be useful for transfecting cells with nucleic acid encoding the CaMKK2 protein, which the transformed host cell is cultured and maintained under conditions wherein expression of the CaMKK2 protein takes place. The genetic construct may be transformed or transduced into a cell. The genetic construct may be formulated into any suitable type of delivery vehicle including, for example, a viral vector, lentiviral expression, mRNA electroporation, and lipid-mediated transfection for delivery into a cell. The genetic construct may be part of the genetic material in attenuated live microorganisms or recombinant microbial vectors which live in cells. The genetic construct may be present in the cell as a functioning extrachromosomal molecule.

[0096] Further provided herein is a cell transformed or transduced with a system or component thereof as detailed herein. The cell may be an immune cell. In some embodiments, the cell is an NK cell. a. Viral Vectors

[0097] The genetic construct may be a viral vector. Further provided herein is a viral delivery system. Viral delivery systems may include, for example, lentivirus, retrovirus, adenovirus, mRNA electroporation, or nanoparticles. In some embodiments, the vector is aDocket No.028193-0031-WO01 / 7879 modified lentiviral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. The AAV vector is a small virus belonging to the genus Dependovirus of the Parvoviridae family that infects humans and some other primate species.

[0098] AAV vectors may be used to deliver polynucleotides encoding a CaMKK2 protein using various construct configurations. For example, AAV vectors may deliver different CaMKK2 protein expression cassettes on separate vectors or on the same vector. Alternatively, different CaMKK2 protein expression cassettes may be combined in a single AAV vector. In some embodiments, the AAV vector has a 4.7 kb packaging limit.

[0099] In some embodiments, the AAV vector is a modified AAV vector. The modified AAV vector may be capable of delivering and expressing the CaMKK2 protein in the cell of a mammal. For example, the modified AAV vector may be an AAV-SASTG vector (Piacentino et al. Human Gene Therapy 2012, 23, 635–646). The modified AAV vector may be based on one or more of several capsid types, including AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. The modified AAV vector may be based on AAV2 pseudotype with alternative muscle-tropic AAV capsids, such as AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5, and AAV / SASTG vectors that efficiently transduce skeletal muscle or cardiac muscle by systemic and local delivery (Seto et al. Current Gene Therapy 2012, 12, 139-151). The modified AAV vector may be AAV2i8G9 (Shen et al. J. Biol. Chem.2013, 288, 28814- 28823). [000100] The genetic construct may comprise a polynucleotide sequence of SEQ ID NO: 2, 3, 5, or 6, or a combination thereof or a complement thereof. The genetic construct may encode a polypeptide sequence of SEQ ID NO: 1 or 4 or a combination thereof. 4. Additional Therapies [000101] In some embodiments, the compositions and methods detailed herein further include additional therapies. As used herein, the term “standard of care treatment” or “additional therapy” or “additional treatment” are used interchangeably and refer to any other standard cancer treatments / additional cancer treatments that do not include the specific compositions detailed herein for increasing CaMKK2 in a NK cell. Additional cancer therapies may comprise a small molecule, peptide, polypeptide, antibody, nucleotide, polynucleotide, lipid, or carbohydrate, or a combination thereof. Additional cancer therapies may be synthesized and / or extracted and / or purified by any suitable means known in the art. Additional cancer therapies may be commercially available. Additional cancer therapies may include, for example, chemotherapy, immunotherapy, radiation therapy, hormone therapy,Docket No.028193-0031-WO01 / 7879 targeted drug therapy, cryoablation, antibody drug conjugates, and surgery, or a combination thereof. Hormone therapy, for example, may block hormone synthesis such as blocking estrogen synthesis. An effective amount of the additional therapy may be administered. [000102] Chemotherapy may include, for example, an antimitotic agent, an alkylating agent, an antimetabolite, an antimicrotubule agent, a topoisomerase inhibitor, a cytotoxic agent, a cell cycle inhibitor, a growth factor inhibitor, a histone deacetylase (HDAC) inhibitor, and an inhibitor of a pathway that cross-talks with and activates ER transcriptional activity, or a combination thereof. [000103] Alkylating agents may include, for example, cisplatin (PLATINOL®), oxaliplatin (ELOXATIN®), chlorambucil (LEUKERAN®), procarbazine (MATULANE®; NATULAN®), or carmustine (BiCNU®), or a combination thereof. Antimetabolites may include, for example, methotrexate (also known as amethopterin), 5-fluorouracil, cytarabine (also known as cytosine arabinoside or ara-C; CYTOSAR®), or gemcitabine (GEMZAR®), or a combination thereof. Antimicrotubule agents may include, for example, vinblastine (VELBAN®; VELBE®), or paclitaxel (TAXOL®), or a combination thereof. Topoisomerase inhibitors may include, for example, etoposide (VEPESID®), or doxorubicin (ADRIAMYCIN®; MYOCET®), or a combination thereof. Cytotoxic agents may include, for example, bleomycin (BLENOXANE®). Growth factor inhibitors may include, for example, human epidermal growth factor receptor 2 (HER2) inhibitors. HER2 inhibitors include, for example, trastuzumab (HERCEPTIN®), deruxtecan, sacitizumab, and / or ado-trastuzumab emtansine (KADCYLA®). HDAC inhibitors may include, for example, vorinostat (ZOLINZA®), romidepsin (ISTODAX®), chidamide (also known as tucidinostat; EPIDAZA®; HIYASTA™), panobinostat (FARYDAK®), belinostat (also known as BELEODAQ® or PXD101), valproic acid (DEPAKOTE®; DEPAKENE®; STAVZOR®)), mocetinostat (also known as MGCD0103), abexinostat (also known as PCI-24781), entinostat (also known as SNDX-275 or MS-275), pracinostat (also known as SB939), resminostat (also known as 4SC-201 or RAS2410), givinostat (also known as gavinostat or ITF2357), quisinostat (also known as JNJ-26481585), kevetrin, CUDC-101, AR-42, tefinostat (also known as CHR-2845), nanatinostat (also known as CHR-3996), domatinostat (also known as 4SC-202), ivaltinostat (also known as CG-200745), rocilinostat (also known as ACY-1215), or sulforaphane, or a combination thereof. Inhibitors of a pathway that cross-talks with and activates ER transcriptional activity may include, for example, a phosphoinositide 3-kinase (PI3K) inhibitor, a heat shock protein 90 (HSP90) inhibitor, or a mammalian target of rapamycin (mTOR) inhibitor. mTOR inhibitors include, for example, everolimus (AFINITOR®; VOTUBIA®; ZORTRESS®). In some embodiments, the HDAC inhibitor comprisesDocket No.028193-0031-WO01 / 7879 vorinostat (ZOLINZA®). In some embodiments, the HDAC inhibitor comprises romidepsin (ISTODAX®). [000104] Immunotherapies may include, for example, a checkpoint inhibitor, or denosumab (PROLIA®; XGEVA®), or a combination thereof. “Checkpoint inhibitor” or “immune checkpoint inhibitor” may also be referred to as an immune checkpoint blockade (ICB) therapy. Checkpoint inhibitors may comprise an antibody. Checkpoint inhibitors may include, for example, an antibody to programmed cell death protein 1 (PD1) (anti-PD1), or an antibody to cytotoxic T-lymphocyte-associated protein 4 (CTLA4) (anti-CTLA4), or an antibody to programmed death-ligand 1 (PDL1) (anti-PDL1), or DMXAA (sting agonist; also known as ASA404, vadimezan, or dimethylxanthone acetic acid) or a combination thereof. “Anti-PD1” refers to an antibody that binds PD1, “anti-CTLA4” refers to an antibody that binds CTLA4, and “anti-PDL1” refers to an antibody that binds PDL1. In some embodiments, the PD-1 antibody comprises pembrolizumab (KEYTRUDA®) or nivolumab (OPDIVOo®). In some embodiments, the CTLA-4 antibody comprises ipilimumab (YERVOY®). [000105] Antibody drug conjugates may include, for example, gemtuzumab ozogamicin (MYLOTARG™), brentuximab vedotin (ADCETRIS®), ado-trastuzumab emtansine (KADCYLA®), inotuzumab ozogamicin (BESPONSA®), polatuzumab vedotin (POLIVY®), enfortumab vedotin (PADCEV®), fam-trastuzumab deruxtecan (ENHERTU®), sacituzumab govitecan (TRODELVY®), loncastuximab tesirine (ZYNLONTA®), tisotumab vedotin (TIVDAK®), mirvetuximab soravtansinegynx (ELAHERE™), moxetumomab pasudotox (LUMOXITI™), belantamab mafodotin-blmf (BLENREP®), cetuximab saratolacan (AKALUX®), or disitamab vedotin (AIDIXI®), or a combination thereof. 5. Pharmaceutical Compositions [000106] Further provided herein are pharmaceutical compositions comprising the above- described modified NK cell or genetic constructs. In some embodiments, the pharmaceutical composition may comprise about 1 ng to about 10 mg of DNA encoding the CaMKK2 protein. The modified NK cell or genetic constructs as detailed herein, or at least one component thereof, may be formulated into pharmaceutical compositions in accordance with standard techniques well known to those skilled in the pharmaceutical art. The pharmaceutical compositions can be formulated according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen free, and particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity may include sodium chloride, dextrose,Docket No.028193-0031-WO01 / 7879 mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. [000107] The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules as vehicles, adjuvants, carriers, or diluents. The term “pharmaceutically acceptable carrier,” may be a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Pharmaceutically acceptable carriers include, for example, diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, emollients, propellants, humectants, powders, pH adjusting agents, and combinations thereof. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent may be a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. The transfection facilitating agent may be poly-L- glutamate, and more preferably, the poly-L-glutamate may be present in the composition for gene editing in skeletal muscle or cardiac muscle at a concentration less than 6 mg / mL. 6. Administration [000108] The genetic constructs as detailed herein, or at least one component thereof, may be administered or delivered to a cell. Methods of introducing a nucleic acid into a host cell are known in the art, and any known method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, polycation or lipid:nucleic acid conjugates, lipofection, electroporation, nucleofection, immunoliposomes, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE- dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, and the like. In some embodiments, the composition may be delivered by mRNA delivery and ribonucleoprotein (RNP) complex delivery. The system, genetic construct, or composition comprising the same, may be electroporated using BioRad Gene Pulser Xcell or Amaxa Nucleofector IIb devices or other electroporation device. Several different buffers may be used, including BioRad electroporation solution, Sigma phosphate-buffered salineDocket No.028193-0031-WO01 / 7879 product #D8537 (PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V (N.V.). Transfections may include a transfection reagent, such as Lipofectamine 2000. [000109] The modified NK cell or genetic construct as detailed herein, or at least one component thereof, or the pharmaceutical compositions comprising the same, may be administered to a subject or cell. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration. The presently disclosed modified NK cell or genetic constructs or compositions comprising the same, may be administered to a subject by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, intranasal, intravaginal, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intradermally, epidermally, intramuscular, intranasal, intrathecal, intracranial, and intraarticular or combinations thereof. In certain embodiments, the genetic construct, or composition comprising the same, is administered to a subject intramuscularly, intravenously, or a combination thereof. The modified NK cell or genetic construct or compositions comprising the same may be delivered to a subject by several technologies including DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome mediated, nanoparticle facilitated, recombinant vectors such as recombinant lentivirus, recombinant adenovirus, and recombinant adenovirus associated virus. The composition may be injected into the brain or other component of the central nervous system. The composition may be injected into the skeletal muscle or cardiac muscle. For example, the composition may be injected into the tibialis anterior muscle or tail. For veterinary use, the modified NK cell or genetic construct or compositions comprising the same may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian may readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The modified NK cell or genetic construct or compositions comprising the same may be administered by traditional syringes, needleless injection devices, “microprojectile bombardment gone guns,” or other physical methods such as electroporation (“EP”), “hydrodynamic method”, or ultrasound. Alternatively, transient in vivo delivery of polynucleotides encoding CaMKK2 protein by non-viral or non-integrating viral gene transfer, or by direct delivery of purified proteins and gRNAs containing cell-penetrating motifs may enable highly specific correction and / or restoration in situ with minimal or no risk of exogenous DNA integration.Docket No.028193-0031-WO01 / 7879 [000110] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated. The modified NK cell or genetic construct or polynucleotides encoding CaMKK2 protein and the at least one additional therapy may be administered together or simultaneously, they may be administered at different times or sequentially. [000111] The modified NK cell or genetic construct, with or without the at least one additional therapy, may be administered to the subject once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months. The modified NK cell or genetic construct may be administered to the subject for 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years. In some embodiments, the modified NK cell or genetic construct, with or without the at least one additional therapy, is administered to the subject until the subject's cancer progresses on therapy. [000112] In some embodiments, the modified NK cell or genetic construct is administered to the subject once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months. In some embodiments, the modified NK cell or genetic construct is administered to the subject for 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years. [000113] Upon delivery of the presently disclosed modified NK cell or genetic construct as detailed herein, or at least one component thereof, or the pharmaceutical compositions comprising the same, and thereupon the vector into the cells of the subject, the transfected cells may express the CaMKK2 protein. 7. Kits [000114] Provided herein is a kit, which may be used to increase the survival and / or proliferation of a NK cell in a tumor of a subject. The kit comprises genetic constructs or a composition comprising the same, for increasing the survival and / or proliferation of a NK cell in a tumor of a subject, as described above, and instructions for using said composition. InDocket No.028193-0031-WO01 / 7879 some embodiments, the kit comprises at least one CaMKK2 protein comprising a polypeptide of SEQ ID NO: 1 and / or a polynucleotide sequence comprising SEQ ID NO: 2 or 3, a complement thereof, a variant thereof, or fragment thereof. In some embodiments, the kit comprises at least one LDH protein comprising a polypeptide of SEQ ID NO: 4 and / or a polynucleotide sequence of SEQ ID NO: 5 or 6, a complement thereof, a variant thereof, or fragment thereof. The kit may further include instructions for using the compositions or genetic constructs. [000115] Instructions included in kits may be affixed to packaging material or may be included as a package insert. While the instructions are typically written on printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” may include the address of an internet site that provides the instructions. 8. Methods a. Methods of Increasing Survival and / or Proliferation of a NK Cell [000116] Provided herein are methods of increasing survival and / or proliferation of a NK cell in a tumor of a subject. The methods may include isolating the NK cell from a biological sample from the subject to generate an isolated NK cell, administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell, and administering to the subject the modified NK cell. In some embodiments, method further comprises administering a polynucleotide encoding LDH to the isolated or modified NK cell to increase the level of CaMKK2 in the NK cell. [000117] In some embodiments, the administration of the modified NK cell decreases the rate of progression of tumor growth in the subject, as compared to the rate of progression of tumor growth prior to the modified NK cell treatment. In some embodiments, the administration of the modified NK cell decreases the rate of cancer metastasis in the subject, as compared to the rate of cancer metastasis prior to the modified NK cell treatment. In some embodiments, the administration of the modified NK cell decreases the size of a tumor in the subject, as compared to the size of the tumor prior to the modified NK cell treatment. The rate of progression of tumor growth, and / or the rate of cancer metastasis, and / or the size of the tumor may be decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,Docket No.028193-0031-WO01 / 7879 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The rate of progression of tumor growth, and / or the rate of cancer metastasis, and / or the size of the tumor may be decreased by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The rate of progression of tumor growth, and / or the rate of cancer metastasis, and / or the size of the tumor may be decreased by about 5-95%, 10-90%, 15-85%, 20-80%, or 1.5-fold to 10-fold, relative to a control. [000118] In some embodiments, the modified NK cell is administered to the subject once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months. In some embodiments, the modified NK cell is administered to the subject for 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years. [000119] In some embodiments, the method further comprises administering to the subject at least one additional therapy. b. Methods of Increasing the Level of CaMKK2 in a NK Cell [000120] Provided herein are methods of increasing the level of CaMKK2 in a NK cell in a tumor of a subject. The methods may include isolating the NK cell from a biological sample from the subject to generate an isolated NK cell, administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell, and administering to the subject the modified NK cell. In some embodiments, method further comprises administering a polynucleotide encoding LDH to the isolated or modified NK cell to increase the level of CaMKK2 in the NK cell. [000121] In some embodiments, the administration of the modified NK cell decreases the rate of progression of tumor growth in the subject, as compared to the rate of progression of tumor growth prior to the modified NK cell treatment. In some embodiments, the administration of the modified NK cell decreases the rate of cancer metastasis in the subject, as compared to the rate of cancer metastasis prior to the modified NK cell treatment. InDocket No.028193-0031-WO01 / 7879 some embodiments, the administration of the modified NK cell decreases the size of a tumor in the subject, as compared to the size of the tumor prior to the modified NK cell treatment. The rate of progression of tumor growth, and / or the rate of cancer metastasis, and / or the size of the tumor may be decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The rate of progression of tumor growth, and / or the rate of cancer metastasis, and / or the size of the tumor may be decreased by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The rate of progression of tumor growth, and / or the rate of cancer metastasis, and / or the size of the tumor may be decreased by about 5-95%, 10-90%, 15-85%, 20-80%, or 1.5-fold to 10-fold, relative to a control. [000122] In some embodiments, the modified NK cell is administered to the subject once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months. In some embodiments, the modified NK cell is administered to the subject for 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years. [000123] In some embodiments, the method further comprises administering to the subject at least one additional therapy. c. Methods of Treating Cancer [000124] Provided herein are methods of treating cancer in a subject. The methods may include isolating the NK cell from a biological sample from the subject to generate an isolated NK cell, administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell, and administering to the subject the modified NK cell. In some embodiments, method further comprises administering a polynucleotide encoding LDH to the isolated or modified NK cell to increase the level of CaMKK2 in the NK cell. [000125] In some embodiments, the administration of the modified NK cell decreases the rate of progression of tumor growth in the subject, as compared to the rate of progression ofDocket No.028193-0031-WO01 / 7879 tumor growth prior to the modified NK cell treatment. In some embodiments, the administration of the modified NK cell decreases the rate of cancer metastasis in the subject, as compared to the rate of cancer metastasis prior to the modified NK cell treatment. In some embodiments, the administration of the modified NK cell decreases the size of a tumor in the subject, as compared to the size of the tumor prior to the modified NK cell treatment. The rate of progression of tumor growth, and / or the rate of cancer metastasis, and / or the size of the tumor may be decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The rate of progression of tumor growth, and / or the rate of cancer metastasis, and / or the size of the tumor may be decreased by less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control. The rate of progression of tumor growth, and / or the rate of cancer metastasis, and / or the size of the tumor may be decreased by about 5-95%, 10-90%, 15-85%, 20-80%, or 1.5-fold to 10-fold, relative to a control. [000126] In some embodiments, the modified NK cell is administered to the subject once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months. In some embodiments, the modified NK cell is administered to the subject for 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years. [000127] In some embodiments, the method further comprises administering to the subject at least one additional therapy. [000128] In some embodiments, the compositions and methods detailed herein may be used against solid tumors. The solid tumors may have a hypoxic, nutrient-depleted, immune suppressive tumor microenvironment. The solid cancer may include, for example, the solid cancer comprises melanoma, colon cancer, breast cancer, lung cancer, head and neck cancer, laryngeal cancer, oral cancer, esophageal cancer, gastric cancer, stomach cancer, colon cancer, bladder cancer, liver cancer, pancreatic cancer, kidney cancer, brain cancer, bone cancer, glioblastoma, sarcoma, ovarian cancer, endometrial cancer, or prostate cancer, or a combination thereof.Docket No.028193-0031-WO01 / 7879 9. Examples [000129] The foregoing may be better understood by reference to the following examples, which are presented for purposes of illustration and are not intended to limit the scope of the invention. The present disclosure has multiple aspects and embodiments, illustrated by the appended non-limiting examples. Example 1 Materials and Methods [000130] Abbreviations: ĮCHC Į-cyano-4-hydroxycinnamic acid AMPK AMP-activated protein kinase BMDM Bone marrow-derived macrophage CaMKK2 Calcium / calmodulin-dependent protein kinase kinase 2 CCL4 Chemokine ligands 4 CCR5 C-C chemokine receptor type 5 CFSE Carboxyfluorescein succinimidyl ester CRISPR Clustered regularly interspaced short palindromic repeats DN Double negative eGFP Enhanced green fluorescent protein GPCR G protein-coupled receptor HDAC Histone deacetylase HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIF1Į Hypoxia-inducible factor 1Į IL2 Interleukin 2 IL15 Interleukin 15 IVIS In vivo imaging system KIR Killer immunoglobulin-like receptor KO Knock-out LDD Ligand-directed degrader LKB1 Live kinase B1 MCT Monocarboxylate transporter mTOR Mammalian target of rapamycin NCR Natural cytotoxicity receptor NK Natural killer cellDocket No.028193-0031-WO01 / 7879 NKT Natural killer T cell NM Normal media NSG NOD.Cg-Prkdc^ಠƍ^ Il2rg^ஆ¹^^^ / SzJ Nu / nu athymic nude PIPES Piperazine-N,Nƍ-bis (2-ethanesulfonic acid) TAM Tumor-associated macrophage TCM Tumor-conditioned media TGF-ȕ Transforming growth factor beta UMAP Uniform Manifold Approximation and Projection [000131] Isolation and Culture of Primary NK Cells. Spleens from WT or CaMKK2 KO BL / 6 female mice aged 10-12 weeks were harvested and crushed through a 40 ^m cell strainer into PBS + 2% FBS. The EasySep Mouse NK Cell Isolation Kit (StemCell Technologies, cat no.19855) was used to isolate NK cells through negative selection. NK cell purity was measured using an Accuri C6 flow cytometer (BD Biosciences) to determine the percentage of purified cells expressing CD49b. The following antibodies were used: CD49b-PE (BioLegend, cat no.103506) and isotype control (Invitrogen, cat no.12-4888-81). NK cells were cultured in either a 50 / 50 mixture of EO771 TCM and normal growth media, or normal growth media alone. Normal growth media consists of RMPI 1640 containing 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM HEPES, 2 mM L-glutamine, 55 ^M 2-mercaptoethanol, and 100 units / mL Penicillin+ 100 ^g / mL Streptomycin. Experiments requiring pH stabilization used 25 mM HEPES and 25 mM PIPES in place of 10 mM HEPES. All reagents were purchased from Gibco. 100 ng / mL IL2 (PeproTech, cat no.212-12) and / or 100 ng / mL IL15 (PeproTech, cat no.210-15) were added to the culture media depending on the experiment. All cells were incubated in a humidified chamber at 37°C in a 5% CO2 atmosphere. [000132] Cancer Cell Lines and Growth Media. EO771 is a murine breast tumor line acquired from the lab of Mark Dewhirst (Duke University). A7C11 is a murine breast tumor line acquired from the lab of Jose Conejo-Garcia (Moffitt Cancer Center). 4T1 is a murine breast tumor line purchase from ATCC. BT-474 is a human breast tumor line also purchased from ATCC. EO771, A7C11, 4T1, and BT474 were cultured in RPMI 1640 containing 10% FBS, 1 mM sodium pyruvate (NaPy), and 0.1 mM non-essential amino acids (NEAA). B16-F10 is a lung-tropic murine melanoma line purchased from the Duke Cell Culture Facility. YUMM1.7 is a mouse melanoma line purchased from ATCC. MDA-231 is a human breast cancer line also purchased from ATCC. B16-F10, YUMM1.7, and MDA-231 were cultured in DMEM containing 10% FBS, 1 mM NaPy, and 0.1 mM NEAAs. PY230 is aDocket No.028193-0031-WO01 / 7879 murine breast tumor line purchased from ATCC and cultured in F-12K Nutrient Mixture, Kaighn’s Modification (Gibco, cat no. 21127-022) containing 5% FBS and 0.1% of MITO serum extender. TamR is a human breast tumor line derived from MCF7 cells (ATCC) through prolonged exposure to tamoxifen. TamR cells were cultured in DMEM / F12 (1:1) Ham media (Gibco, cat no.11330-032) containing 10% FBS, 1 mM NaPy, 0.1 mM NEAAs, and 100 nM 4-OH-tamoxifen. NK-92 is a human NK cell lymphoma line purchased from the Duke Cell Culture Facility. These cells were grown in suspension in Minimum Essential Medium (Sigma, cat no. M4526) containing 12.5% FBS, 12.5% horse Serum, 0.2 mM myo- inositol, 0.02 mM folic acid, 2 mM L-glutamine, 0.1 mM ȕ-Mercaptoethanol, and 5,000 units / mL human IL2 (BD Bioscience, cat no.40043B). All cells were incubated in a humidified chamber at 37 °C in a 5% CO2atmosphere. [000133] Generation of Tumor-Conditioned Media. One million EO771 or A7C11 tumor cells were plated on a 10-cm cell culture dish in their standard RMPI growth media and incubated at 37°C. After exactly 72 hours, the growth media was recovered from each plate, centrifuged at 2000 x g for 5 minutes to remove debris, and the supernatant was frozen at í80°C. [000134] Mouse Strains. The CaMKK2í / í(KO) mouse strain was originally generated in the lab of Anthony Means (Baylor College of Medicine). These mice were backcrossed for five generations to WT C57BL / 6 mice (Jackson Labs) and have a genetic background that is at least 95% C57BL / 6. The [Tg(CaMKK2-eGFP)BL / 6] reporter mouse line originates from the Mutant Mouse Regional Resource Center. This strain features the sequence of enhanced green fluorescent protein (eGFP), followed by a polyadenylation sequence, inserted into the mouse genomic bacterial artificial chromosome RP23-31J24 at the initiation codon of the CaMKK2 gene. The Racioppi lab had backcrossed the original strain to Jackson Lab’s WT BL / 6 mice for 10 generations, generating the mice used in the studies. [000135] NKp46-iCre mice were acquired from the lab of Joseph Sun (Memorial Sloan Kettering Cancer Center) and were originally created by Eric Vivier (Centre d’Immunologie de Marseille-Luminy). This strain features the Cre coding sequence preceded by an IRES inserted into the 3’UTR of the NKp46 (Ncr1) sequence. These mice were bred with CaMKK2 flox / flox mice to generate Ncr1-cre+ / íCaMKK2 flox / flox experimental mice and Ncr1-cre+ / íCaMKK2wt / wtcontrols. The CaMKK2 flox mice were originally generated by the lab of Anthony Means (Baylor College of Medicine). [000136] Tamoxifen-inducible ERT2-cre+ / íCaMKK2flox / floxmice (including ERT2-negative controls) were generated by breeding CaMKK2flox / floxmice (described above) with UBC-Cre-Docket No.028193-0031-WO01 / 7879 ERT2 mice acquired from Jackson Labs (strain #007001). ERT2+mice and ERT2ícontrols were dosed with 2 mg tamoxifen citrate (Sigma-Aldrich, cat no. T9262) in corn oil via oral gavage daily for one week, and splenic NK cells were harvested 5 days after dosing ended. [000137] The following mice were purchased from Jackson Labs as needed: WT C57BL / 6 females (strain # 000664), NSG females (strain # 005557), and athymic nude females, or nu / nu (strain # 002019). [000138] CFSE Proliferation Assay. NK cell proliferation was measured via dilution of the anime reactive CFSE dye. Splenic NK cells were treated with 5 ^M CellTrace CFSE (Invitrogen, cat no. C34554A) in RMPI media + 5% FBS for 5 minutes, washed twice with the same media, and cultured under various conditions for 72 hours. CFSE fluorescence decreases by half with each round of cell division, enabling us to identify the proliferating fraction of NK cells via flow cytometry. NK cells were gated as live CD45+ / CD3í / NK1.1+ / CD49b+cells. [000139] In vitro Migration Assay. NK cell migration was measured using a transwell migration assay. Splenic NK cells that had been cultured under various conditions for 72 hours were counted, and 500,000 live NK cells were suspended in 100 μL of normal growth media and added to the upper insert of a transwell migration chamber with 5 μm pores (Corning, cat no. 3421). 600 μL of normal growth media containing 0 or 250 ng / mL CCL4 (BioLegend, cat no. 554602) was added to the lower chamber, and the migration plate was incubated at 37 °C for 3 hours. The Accuri C6 flow cytometer (BD Biosciences) was used to count the total number of CD49b+cells present in the lower chamber. [000140] In vitro NK Cytotoxicity Assay. NK cell cytotoxic activity was measured via a co-culture with YAC1 tumor cells. YAC1 cells, a canonical MHC I-negative target for NK cells, were cultured in RMPI 1640 containing 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES, and 3.5 g / L D-(+)-glucose (Sigma). The YAC1 cells were stained with CellTrace CFSE (Invitrogen, cat no. C34554A) for quick identification on flow cytometry. YAC1 cells were counted using a microscope hemocytometer chamber (Hausser Scientific), and 25,000 cells were added to each internal well of a flat-bottom 96 well plate (Corning) in 75 ^L of their growth media. Splenic NK cells that had been cultured for 72 hours under various conditions were counted, and live NK cells were added to the YAC-1 cells at a ratio of 1:25, 1:5, 1:1, and 5:1 (NK:YAC1). The volume of each co-culture well was topped up to 150 ^L with NK growth media. Three biological replicates were carried out at each co-culture ratio. The NK cells and YAC1 cells were incubated at 37°C for 4 hours, and cells were then stained with Live / dead stain and anti-CD45-BV605 for flow cytometry analysis. YAC1 cellsDocket No.028193-0031-WO01 / 7879 were identified as CD45í / CFSE+. The dead fraction of YAC1 cells (positive live / dead staining) were represented as a fraction of total YAC1 cells. [000141] Flow Cytometry. The following reagents were used to prepare NK cells for flow cytometry. Anti-CD16 / 32 (Invitrogen, cat no.14-0161-85) and anti-CD16.2 (BioLegend, cat no.149502) were used for Fc receptor blocking, and LIVE / DEAD Fixable Violet Dead Cell Stain (Invitrogen, cat no. L34964) was used as a viability stain. NK cells were then stained with a combination of cell-surface fluorophore-antibody conjugates in BD Horizon Brilliant Stain, as listed in Table 1. NK cells were then permeabilized using eBioscience Fixation / Perm solution (Invitrogen, cat no. 00-5123-43 and 00-5223-56) and stained with some combination of intracellular fluorophore-antibody conjugates in Permeabilization Buffer (Invitrogen, cat no. 00-8333-56). Cells were then fixed in BD Cytofix fixation buffer diluted 1:4 (BD Bioscience, cat no. 554655). Flow cytometry data was collected on an LSRFortessa X-20 (BD Biosciences) and analyzed using FlowJo software version 10.7.1.Docket No.028193-0031-WO01 / 7879[000142] Western Immunoblotting. The primary antibodies listed in Table 2 were used to detect the indicated proteins through Western immunoblotting. Fluorescent secondary antibodies purchased from Biotium were used for LI-COR imaging. Western blots were scanned using a LI-COR Odyssey Classic fluorescence scanner and processed using LI- COR Image Studio software, version 4.0.[000143] Cell pellets from the source indicated in the corresponding ¿gure legend were suspended in RIPA buffer (Table 3), Àash-frozen in liquid nitrogen, and rotated at 4 °C for 30 minutes.Docket No.028193-0031-WO01 / 7879[000144] 40 mg of protein per sample was resolved on a 7.5% acrylamide gel using SDS- PAGE and transferred onto a nitrocellulose membrane (LI-COR, cat no. 926–31092). The nitrocellulose membrane was blocked with 1 g / L casein hydrolysate (MP Bio, cat no. 02101290) and 4.5 g / L ¿sh gelatin (Sigma-Aldrich, cat no. G7041–100G) and then incubated with the primary antibodies listed in Table 2. Images were captured using the LI- COR Odyssey Classic scanner and processed using LI-COR Image Studio software, version 4.0. [000145] Creation of Plasmid Vector to Knock-Out MHC class I. The EO771 cell line was engineered using CRISPR to knock-out both alleles of the ȕ2m locus, eliminating expression of the major histocompatibility complex class I. The Lenti-Cas9-gRNA-GFP plasmid vector (Addgene plasmid #124770) was first modified to contain two specific guide RNA (gRNA) sequences targeting the mouse ȕ2m locus. This plasmid was linearized via digestion with the BsmBI restriction enzyme, separated on a 1% agarose gel, and recovered using the QIAquick gel extraction kit (Qiagen, cat no. 28706X4). The following forward and reverse primer sets were annealed together through gradual cooling from 95 °C and then incubated with the linearized plasmid at room temperature for 30 minutes in the presence of the T4 ligase: B2M-1F: 5’-CACCGAGTATACTCACGCCACCCAC-3’ (SEQ ID NO: 7)Docket No.028193-0031-WO01 / 7879 B2M-1R: 5’-AAACGTGGGTGGCGTGAGTATACTC-3’ (SEQ ID NO: 8) B2M-2F: 5’-CACCGAGTCGTCAGCATGGCTCGCT-3’ (SEQ ID NO: 9) B2M-2R: 5’-AAACAGCGAGCCATGCTGACGACTC-3’ (SEQ ID NO: 10) [000146] 5 μg of each ligated plasmid was incubated with 50 ^L of Stbl3 homologous recombination-deficient bacteria (ThermoFisher, cat no. 737303) on ice for 30 minutes. The bacteria were then heat shocked at 42 °C for 40 seconds, added to 300 ^L of SOC media, shaken at 37 degrees for 1 hour, and plated onto LB-Amp plates (100 ^g / mL ampicillin). The two plates were incubated for 24 hours at 37 degrees, and 5 colonies from each plate were picked for a 24-hour expansion in 3 mL liquid LB-Amp media. The QIAprep mini prep kit (Qiagen, cat no. 27106X4) was used to isolated plasmid DNA from the 10 expanded bacteria colonies, and the samples were submitted to Eton Bio for sequencing with the “hU6” universal primer set. Using the procedure described in A.11, EO771 cells were transfected with the plasmid-cas9-gRNA constructs described above, and EO771 cells with homozygous ablation of ȕ2m were isolated using flow cytometry-based cell sorting to select for GFP+, MHC I-negative cells. [000147] Transfection of Cell Lines. Various cell lines were modified to express a Tet- inducible shRNA knock-down construct or the luciferase enzyme, which enables bioluminescence imaging of tumor burden. To generate lentivirus transfection vectors, HEK293T cells (ATCC) were grown to 40% confluency in DMEM containing 10% FBS, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 2 mM L-glutamine. The following transfection mixture was prepared for one 10 cm culture plate: 263 Opti-Mem, 17 ^L FuGENE-6 (Promega, cat no. E2692), 280 ng VSVG envelope vector (Addgene plasmid #8454), 2.8 ^g PsPAX2 Lentivirus packaging vector (Addgene plasmid #12260), and 2.8 μg of the transfection vector (e.g., luciferase, Tet-inducible shRNA, or Cas9 / ȕ2mg-RNA). The transfection mixture was incubated at room temperature for 30 minutes, added dropwise to the HEK293 cells, and the plate was incubated at 37°C. 24 hours later, the media was aspirated and replaced with the standard DMEM growth media containing 30% FBS. 24 hours later, the lentivirus-containing media was recovered, filtered through a 45 ^m filter, and frozen at í80°C. New DMEM growth media containing 30% FBS was added to the plates, and this media was recovered and filtered in the same manner 24 hours later. [000148] B16-F10 or EO771 cells were grown to 70% confluency in their standard growth media. The lentivirus-containing media was diluted in an equal volume of tumor growth media and added to the B16-F10 or EO771 cells. 24 hours later, the first round of lentivirus-Docket No.028193-0031-WO01 / 7879 containing media was aspirated, and a second round of diluted lentivirus-containing media was added. 24 hours later, the virus-containing media was replaced with standard tumor growth media, and the cells were incubated for 48 hours. Transfected cells were split across a six-well plate and cultured with various concentrations of neomycin (Geniticin brand, Gibco, cat no. 3480) ranging from 50 ^g / mL to 2000 ^g / mL for 10 days. The highest neomycin concentration that failed to kill all tumor cells was chose as the optimal selection dose and expanded. [000149] Tail Vein Metastasis Studies. For NK cell studies, 6-7-week-old female NSG mice were purchased from Jackson Labs and used as models of metastatic tumor growth. The NSG mice were randomized, ear-punched for identification, and their chests shaved to improve bioluminescence signal. Splenic NK cells were isolated from 10-12-week-old WT and CaMKK2 KO female mice and cultured for 24 hours in NK cell growth media containing 100 ng / mL murine IL15. 250,000 live WT or KO NK cells were then injected into the lateral tail vein of NSG mice in a volume of 100 ^L Hank’s Balanced Salt Solution (HBSS, Gibco, cat no. 14025-092). When using the B16-F10 model, 500,000 tumor cells were injected concurrently with the NK adoptive transfer: NK cells and tumor cells were mixed immediately before injection in a total volume of 100 ^L HBSS per mouse. “No NK” control mice were injected with tumor cells alone. Mice were discarded if the intravenous injection was not successful on the first attempt. 200 units of murine IL2 suspended in 100 ^L PBS was injected into the peritoneal cavity of every mouse (including controls) immediately after adoptive transfer of the NK cells and every third day thereafter. When using the ȕ2mí / íEO771 model, 300,000 tumor cells were injected into the lateral tail vein 48 hours prior to adoptive transfer of NK cells to prevent immediate clearance of blood-borne tumor cells. [000150] Starting at day 10 and repeating every 3-4 days thereafter, all mice were anesthetized with isoflurane and administered 100 μL luciferin substrate (Regis Technologies, cat no. 1-360243-200) via retroorbital injection immediately prior to imaging. Luciferase bioluminescence was measured using an IVIS Lumina XR optical imaging system with an exposure time of 2-3 minutes. The signal intensity range was standardized for all images collected in a particular session, and gates were drawn around any signal visualized above the minimum threshold. The total signal intensity from each mouse measured in lumens / sec / cm2was reported as “metastatic burden”. Signals emanating from the tail were treated as primary injection-site tumors and excluded from the “metastatic burden” summation. Studies were not blinded as this would require prohibitive amounts of effort from colleagues otherwise uninvolved in the project.Docket No.028193-0031-WO01 / 7879 [000151] Mice were euthanized and lungs were extracted for flow cytometry profiling of the cellular populations. The lung tissue was mechanically pulverized in 5 mL DMEM + 5% FBS and then enzymatically digested by adding 1 mg / mL Collagenase A (Roche, cat no. 10103586001) and 200 units / mL DNAse I (Sigma, D5025-150KU) and incubated for 30 minutes in a 37 °C shaker. Each sample was then passed through a 40 ^m cell strainer, centrifuged down, and resuspended in 1-2 mL ACK lysis buffer (Gibco, cat no. A10492-01) for 2 minutes to eliminate red blood cells. ACK lysis buffer was diluted in 10 volumes of PBS, and cells were stained with a variety of fluorophore-conjugated antibodies for flow cytometry analysis. [000152] Primary Tumor Studies. 11-12-week-old CaMKK2-eGFP reporter mice were injected subcutaneously with 200,000 E0771 tumor cells in 100 ^L HBSS. Tumor size was measured every 3-4 days using calipers, and upon reaching a volume of 1 cm3, mice were euthanized, and tumors and spleens were extracted. Spleens were crushed through a 40 ^m cell strainer, RBCs were lysed with ACK buffer, and splenocytes were stained for flow cytometry profiling. Tumor cells were mechanically pulverized and digested with Collagenase A and DNAse I, as described in the previous section, and stained for flow cytometry profiling. [000153] Statistical Analysis. Standard error of the mean is shown based on 2-3 biological replicates per experiment. Microsoft Excel was used for statistical analysis. P- value was determined using an unpaired Student’s t-test with a significance threshold of P<0.05 (*P<0.05, **P<0.01, ***P<0.005). Example 2 NK Cells Express Low Basal Levels of CaMKK2 [000154] Immune cells were recovered from eight different tissues in healthy CaMKK2- eGFP reporter mice and gated for ten distinct immune populations or subpopulations in each tissue. The gating strategy is a modified version of a previously published approach used to simultaneously analyze myeloid and lymphoid populations (FIG.3B). Robust expression of the CaMKK2-eGFP reporter was observed in macrophages and monocytes, with expression being low or undetectable in most lymphoid subpopulations (FIG.2A). The only splenic lymphoid population that showed evidence of eGFP expression was a subset of CD11b+NK cells, although eGFP fluorescence levels in these cells were lower than that observed in the monocyte controls (FIG.2A). However, this eGFP+subset represented at least 30% of CD11b+NK cells in every tissue analyzed except the mesenteric lymph nodes (FIG.2B).Docket No.028193-0031-WO01 / 7879 CD11b is a typical myeloid marker, but its expression is also found in NK cells at later maturation stages. The association of CaMKK2 expression with mature effector NK cells was particularly intriguing and warranted further investigation. In contrast, eGFP expression was not observed in CD11bíNK cells, B cells, or any subset of T-cells in any tissues analyzed (FIG.3A), suggesting that mature NK cells are unique among lymphocytes in expressing CaMKK2. Interestingly, a subset of NKT cells also exhibited eGFP expression, especially in the blood and bone marrow (FIG.3A), which reflects the pattern seen in the general NK population. [000155] Another novel finding from this screen was the identification of eGFP in double negative (DN) thymocytes. About half of the DN population was positive for eGFP fluorescence, whereas no other thymocyte subsets, including double and single-positive populations, showed any evidence of expression (FIG.2C). Furthermore, eGFP expression in DN thymocytes was correlated with CD44 (FIG.2C), a marker of the DN1 and DN2 substages. Double negative thymocytes represent the first stage of T-cell development during which unique T-cell receptors are generated and the canonical T-cell marker CD3 is first expressed. Interestingly, the end of the DN2 stage is exactly when VDJ recombination begins, and the loss of CaMKK2 expression at this juncture suggested a possible regulatory role for CaMKK2 in this process. CaMKK2 expression is known to act as a “checkpoint” in the differentiation of various immune progenitor cells, including HSPCs and GMPs in the bone marrow, raising the possibility that CaMKK2 restricts the progression of T-cell progenitors in a similar manner. Example 3 CaMKK2 is Upregulated in NK cells under Tumor Conditions [000156] The initial screen to identify CaMKK2-expressing immune cells was conducted in healthy mice. To explore the effects of the tumor environment on this enzyme, the earlier study was repeated under conditions that mimicked the tumor microenvironment and subsequently in tumor-bearing animals. First, an ex vivo study was performed in which splenic NK cells from CaMKK2íeGFP reporter mice were cultured in tumor-conditioned media (TCM) from EO771 cells. TCM is spent media recovered from tumor cell cultures, and it approximates the chemical composition of the tumor environment. IL2 and IL15 were added to the culture to maintain NK cell viability. Flow cytometry was used to analyze the NK cells 24 hours and 72 hours after plating. In this manner, the surprising observation was made that eGFP fluorescence was dramatically increased at 72 hours, whereas noDocket No.028193-0031-WO01 / 7879 expression was observed at 24 hours (FIG.4A). This was the first data to suggest that a factor in the tumor environment modulates CaMKK2 expression levels. [000157] To validate these results in an animal tumor model, subcutaneous EO771 tumors were propagated in CaMKK2-eGFP reporter mice and harvested for flow cytometry-based immune profiling. Notably, eGFP expression was significantly higher in tumor-infiltrating NK cells than in NK cells from the spleens of the same mice (FIG.4B). Indeed, the level of eGFP fluorescence in the tumor-infiltrating NK cells approached that of macrophages, which served as a positive control (FIG.4C). Moreover, splenic NK cells from tumor-bearing mice did not show an appreciable increase in eGFP expression compared to splenic NK cells from healthy mice (FIG.4D), suggesting that the local tumor environment mediates CaMKK2 upregulation, not systemic factors in tumor-bearing animals. Under basal conditions, CaMKK2 expression was confined to mature CD11b+NK cells, but in the tumor environment, even immature CD11bí / CD27+NK cells were found to express the eGFP reporter, albeit at a lower level than the CD11b+ / CD27ísubset (FIG.4E). Using a panel of markers, including inhibitory KIR receptor Ly49c, activating receptor NKp46, activation marker Granzyme B, and NKT marker CD3, no correlation between NK cell functionality and eGFP expression was identified (FIG.4F). CaMKK2, represented by the eGFP surrogate, was expressed at similar levels across all these subsets. Thus, tumor-associated upregulation of CaMKK2 is not specific to any NK cell subset but occurs broadly throughout the natural killer population, including NKT cells, which are often characterized as a separate cell type. In contrast, T- cells showed no evidence of elevated eGFP expression in the tumor environment (FIG.4C), suggesting that CaMKK2 upregulation is specific to NK cells rather than generalizable across the lymphoid lineage. These findings were repeated in a second independent primary tumor study. [000158] The CaMKK2-eGFP reporter model is a surrogate for transcription as it reflects activity of the CaMKK2 promoter, but a more direct measurement of the enzyme was required to confirm its expression (i.e., Western immunoblots). NK cells are not abundant enough in the tumor environment to use tumor-derived NK cells for immunoblotting. Instead, splenic NK cells were isolated from WT BL / 6 female mice to culture ex vivo in normal growth media (NM) or tumor-conditioned media (TCM). Splenic T-cells were also harvested from WT mice and cultured in the same manner as NK cells. As positive and negative controls for CaMKK2 expression, bone marrow was harvested from WT and CaMKK2í / í(KO) mice and differentiated into bone marrow-derived macrophages (BMDMs) using M-CSF stimulation. The Western immunoblot confirmed robust CaMKK2 expression in the TCM-treated NK cells, but not in the NK cells grown in normal media (FIG.4G), indicating that factors withinDocket No.028193-0031-WO01 / 7879 the tumor context enables CaMKK2 upregulation. T-cells exhibited no CaMKK2 expression under either condition (FIG.4G), which reinforces earlier data and demonstrates that the regulatory mechanisms observed in NK cells are not generalizable among lymphocyte populations. CaMKK2 expression in TCM-treated NK cells was confirmed in a second independent Western immunoblot. Example 4 CaMKK2 Promotes NK Cell Survival and Proliferation In Vitro [000159] Upon establishing that CaMKK2 is expressed in tumor-infiltrating NK cells, the mechanisms by which this enzyme influences NK cell biology using a series of in vitro models were investigated. Two models of CaMKK2 ablation were used in these studies: (1) splenic NK cells harvested from WT and CaMKK2 KO mice, and (2) pharmacologic degradation of CaMKK2 in WT NK cells using a specific, high-affinity CaMKK2 LDD (CC3240). This LDD degrades about 75% of the CaMKK2 protein in primary mouse NK cells (FIG.5D). To approximate tumor conditions in vitro, NK cells were cultured in 50% EO771 TCM. Using this model, the effects of CaMKK2 ablation on a range of NK cell processes were assessed, including apoptosis, proliferation, activation marker expression, direct cytotoxic activity, and migration. [000160] Both the KO and CC3240-treated NK cell groups, when cultured in TCM, exhibit a significant reduction in total live cells compared to the WT and DMSO controls (FIGS.5A- 5E). This reduction in total live NK cells was likely driven by a combination of reduced NK cell survival and reduced proliferation. When cultured in TCM, both KO and CC3240-treated NK cells exhibited reduced viability (FIG.5B; FIG.4F) and elevated levels of the pre- apoptosis marker Annexin V, compared to controls (FIG.5C; FIG.4G). Thus, increased apoptosis is likely the driver of reduced NK cell viability in the CaMKK2-deficient groups, rather than other forms of cell death. A significant reduction in viability was also observed when CC3240-treated NK cells were cultured in A7C11 TCM (FIG.5H), indicating that the pro-survival effects of CaMKK2 are generalizable beyond the EO771 TCM model. Importantly, no changes in viability and Annexin V expression were seen in the CaMKK2- deficient NK cells grown in normal media (FIGS.5A-5C; FIGS.4E-4H), reflecting the necessity of tumor conditions to upregulate CaMKK2. [000161] Next, the effects of CaMKK2 expression on NK cell proliferation were studied. CaMKK2 KO NK cells cultured in TCM exhibited reduced expression of the proliferation marker Ki67 compared to WT NK cells (FIG.6A). To directly measure proliferation, WT NKDocket No.028193-0031-WO01 / 7879 cells were stained with the amine-reactive vital dye CFSE and cultured in TCM with CC3240 or DMSO vehicle. CFSE is diluted with each round of cell division, so reduced CFSE fluorescence serves as a proxy for proliferation. It was observed that the CC3240-treated NK cells were noticeably less likely to undergo division compared to those treated with the DMSO control (FIG.6B). These findings suggest that CaMKK2 expression preserves the proliferative capacity of tumor-conditioned NK cells. As with the survival phenotype, exposure to TCM was required to elicit changes in proliferation upon CaMKK2 ablation (FIGS.6A-6B). [000162] The effects of CaMKK2 expression on in vitro NK cell migration were also assessed. To this end, NK cells were cultured for 72 hours in EO771 TCM with CC3240 or DMSO vehicle, at which point equal numbers of live cells from each group were placed into the upper inserts of a migration chamber containing the CCL4 chemokine in the lower well. After three hours, it was observed that significantly fewer CC3240-treated NK cells had migrated to the bottom chamber compared to the DMSO control (FIG.6C). CCL4-mediated chemotaxis was markedly reduced, but so too was the baseline level of random migration, suggesting a broad dysfunction in the cell movement machinery of CaMKK2-deficient NK cells. WT and KO NK cells express CCR5, the CCL4 receptor, at comparable levels (FIGS. 6D-6E). These data, together with that from the proliferation and cell survival studies, suggested that CaMKK2 expression confers a general fitness advantage on NK cells under the stressful conditions of the tumor environment. [000163] The effects of CaMKK2 expression on the cytotoxic actions of NK cells were assessed through functional marker expression and ex vivo cytotoxicity assays. Flow cytometry profiling of TCM-cultured NK cells showed that CaMKK2 ablation had no effect on the expression of several markers indicative of cytotoxic function, including CD27, NKG2D, and KIR receptor Ly49c (FIG.7A). Granzyme B stained poorly in these cells and is not shown. These results were not surprising given the lack of correlation between eGFP fluorescence and functional marker expression in the CaMKK2 reporter experiments. The direct cytotoxicity of KO or CC3240-treated NK cells was also evaluated. Specifically, NK cells were cultured for 72 hours in TCM with CC3240 or DMSO vehicle, then co-cultured with YAC-1 target cells at various ratios. YAC-1 is an MHC I-negative cell line classically used in NK cell cytotoxicity assays. YAC-1 survival was measured using flow cytometry. In this manner, it was determined that neither pharmacologic ablation of CaMKK2 (FIG.7B) nor genetic ablation (FIG.7C) affects the cytotoxicity of NK cells against YAC1 tumors. Taken together, the in vitro assays showed that CaMKK2 regulates apoptosis, proliferation, andDocket No.028193-0031-WO01 / 7879 migration in tumor-conditioned NK cells, but does not directly affect cytotoxic potency of individual cells. Example 5 CaMKK2 Regulates NK Cell Biology through a Scaffold Mechanism [000164] To study the mechanism by which CaMKK2 regulates NK cell function, the effects of several functionally distinct CaMKK2-modulating compounds were compared. CC8977, YL-36, and GSKi are competitive inhibitors of CaMKK2, whereas the LDDs CC3240 and CC3756 recruit the E3 ubiquitin ligase cereblon to degrade the CaMKK2 protein, mimicking genetic KO of CaMKK2 (currently unpublished). All five compounds are highly specific for CaMKK2 and bind with high affinities. Interestingly, both LDDs but none of the competitive inhibitors reduce viability and Ki67 expression in TCM-cultured NK cells (FIGS.8A-8B), suggesting that the CaMKK2 protein scaffold, but not its kinase function, is important for NK cell regulation. All five compounds reduced AMPK phosphorylation at Thr172, the classic CaMKK2-binding site, confirming that the drugs are active in primary murine NK cells (FIG.8C). These findings rule out the AMPK pathway as the primary mediator of the pro-survival, pro-proliferation phenotype associated with CaMKK2 expression. Rather, the data highlight an important scaffold function for this enzyme in regulating NK cell biology. Tumor-conditioned NK cells were also treated with the most widely used competitive CaMKK2 kinase inhibitor STO609, but this drug was found to reduce NK cell survival even under normal media conditions (FIG.8D), likely reflecting toxicity or lack of specificity at the high concentrations (10 ^M) necessary for CaMKK2 inhibition. [000165] As a primary source of energy and reactive-oxygen species, the mitochondria play an important role in the survival and proliferation of most cell types, including NK cells. The structural integrity, biogenesis, and turnover of mitochondria is tightly regulated as imbalanced mitochondrial activity leads to cellular dysfunction and death. Interestingly, CaMKK2 has been shown to regulate mitochondrial activity in multiple tumor cell lines through modulation of the SDH enzyme complex, raising the possibility that CaMKK2 enhances NK cell fitness through effects on the mitochondria. To explore this possibility, splenic NK cells were cultured in EO771 TCM for 96 hours and treated with a CaMKK2 inhibitor, LDD, or the DMSO vehicle. NK cells were then stained with three fluorescent mitochondrial dyes that were measured using flow cytometry: (1) MitoTracker Green, which stains all mitochondria and measures total mitochondrial mass, (2) MitoMark Red I, a dye whose fluorescence depends on mitochondrial membrane potential, and (3) MitoSox, whichDocket No.028193-0031-WO01 / 7879 measures mitochondrial reactive oxygen species. None of the CaMKK2 inhibitors or LDDs affected total mitochondria mass (FIG.8E), mitochondrial membrane potential (FIG.8F), or production of mitochondrial reactive oxygen species (FIG.8G) in the TCM-treated NK cells. Given these results, mitochondrial regulation was ruled out as the principal mechanism by which CaMKK2 preserves NK cell fitness. Example 6 CaMKK2 expression enhances NK cell Anti-Tumor Immunity [000166] NK cells play many roles in tumor immunity, including detection of de novo tumor formation, clearance of metastatic cells, and recruitment / activation of other immune cells. However, NK cells are a particularly critical component of the anti-metastasis response, and the animal studies focused on this aspect of tumor biology. Changes during the epithelial- mesenchymal transition, which is critical for metastasis, render tumor cells especially vulnerable to NK cell activity. Indeed, NK cells have a significant impact on the dissemination of tumor cells and the growth of metastatic lesions. Conversely, NK cells typically show poor infiltration into primary tumors, and evidence for the role of NK cells in primary tumor progression is mixed. From a translational perspective, metastatic disease is highly relevant as 70% of cancer mortality is attributed to metastasis. For these reasons, the animal studies focused on metastatic disease rather than primary tumor progression. Specifically, intravenous metastasis models were used, in which tumor cells are injected through the lateral tail veins of mice. Although intravenous tumor injection bypasses the early intravasation step, this model preserves the circulation, extravasation, and colonization steps during which NK cells exert the greatest influence. Tail vein models are widely employed in the cancer biology field as a relevant metastasis model. [000167] To limit CaMKK2 ablation to the NK cell compartment, the initial metastasis experiments utilized NSG mice engrafted with NK cells isolated from WT or CaMKK2 KO hosts. NSG mice completely lack endogenous NK cells due to the deletion of the IL-2 receptor Ȗ chain. Thus, NK cells harvested from the spleens of WT or CaMKK2 KO mice can be transfused into NSG hosts in lieu of endogenous NK cells. However, NK cell viability must be maintained by exogenous administration of recombinant IL-2. At the time of transfusion, WT and CaMKK2 KO NK cells were qualitatively similar as measured by the expression of several activation and maturation markers (FIG.9A). To increase tumor cell sensitivity to NK cell activity, the EO771 tumor subline that was employed was engineered to eliminate MHC I expression using a CRISPR-based approach to delete both alleles of ȕ2m (FIG.9B). ȕ2m serves as the common heavy chain of MHC I and is required for theDocket No.028193-0031-WO01 / 7879 stabilization and cell surface expression of the complex. The EO771 cells were also modified to express luciferase, enabling systemic monitoring of tumor burden in live animals using a luciferin-based in vivo imaging system (IVIS). The modified EO771 cells were injected via tail vein, and within two weeks, mice engrafted with CaMKK2 KO NK cells exhibit a significantly larger systemic EO771 metastatic burden than mice engrafted with WT NK cells (FIGS.10A-10C). These results suggested that ablation of CaMKK2 impairs the anti- metastasis and surveillance functions of NK cells. [000168] The metastasis study was repeated using the B16-F10 melanoma model, which expresses low levels of MHC I at baseline (FIG.9B). As observed in the EO771 model, mice engrafted with CaMKK2 KO NK cells exhibited a significantly larger metastatic B16-F10 burden compared to their WT counterparts (FIGS.11A-11B). Post-mortem flow cytometry analysis of the lung tissue, which was the dominant site of metastasis, showed a significant reduction in the prevalence and total number of live NK cells in the lungs of the KO group, likely driven in part by the observed reduction in NK cell viability (FIG.11C). However, the WT and KO groups exhibited no differences in NK cell activation or maturation markers (FIGS.11D-11E), suggesting that NK cell viability, but not cytotoxic function, is compromised upon CaMKK2 ablation. These results reinforced earlier in vitro studies showing reduced viability when CaMKK2 is ablated in TCM-cultured NK cells. [000169] The NK cells transfused in the first set of metastasis studies were harvested from animals with a germline deletion of the CaMKK2 locus, raising the possibility of confounding developmental or compensatory factors. CaMKK2 is expressed in several immune cell precursors in the bone marrow where it regulates lineage commitment and downstream development. CaMKK2 also affects the function of macrophages which in-turn influences NK cells through complex crosstalk interactions. To control for these potential confounders, and to clearly delineate the significance of NK cell-intrinsic functions of CaMKK2, the metastasis experiments were repeated using a tamoxifen-inducible CaMKK2 KO model. This model features constitutive expression of a cre-ERT2 fusion protein that translocates to the nucleus upon tamoxifen induction, excising the floxed CaMKK2 alleles (FIG.12A). Splenic NK cells were harvested from ERT2+and ERT2ícontrols after one week of tamoxifen administration, and the NK cells were co-transfused with luciferase-expressing, GFP+B16 / F10 cells into NSG hosts. Knockdown of CaMKK2 in BMDMs from ERT2+mice, but not in the ERT2ímice, was confirmed by Western immunoblot (FIG.12B). Within two weeks of tumor injection, mice reconstituted with the ERT2+NK cells showed a significantly larger systemic B16-F10 tumor burden compared to the ERT2ícontrols (FIGS. 12C-12D), confirming the importance of CaMKK2 in metastatic progression.Docket No.028193-0031-WO01 / 7879 [000170] Post-mortem flow cytometry analysis showed a dramatic increase in MHC I expression on tumor cells isolated from the lungs of ERT2ímice compared to the ERT2+group (FIG.12E). MHC I expression protects tumors from NK killing, so MHC I upregulation is likely the result of selective pressure exerted on the tumor cells by NK cells. Thus, persistently low MHC I expression on the tumors provided direct evidence of reduced immunoediting when NK cells lose CaMKK2 expression. Flow cytometry profiling of the immune cell repertoire in the lungs showed a reduction in total live NK cells in the ERT2+group compared to that observed in ERT2ímice (FIG.12F), as was observed in prior studies using the germline CaMKK2 KO model. The reduction in live NK cells was likely driven by reduced proliferation, reflected in lower Ki67 expression among ERT2+NK cells, and by a reduction in NK cell viability in the ERT2+group (FIG.12F). [000171] The metastasis studies conducted up to this point had utilized immune deficient animals in which transfused NK cells are the only functional bone marrow-derived immune population. However, many other immune populations are normally present in the tumor environment, and these populations may affect the overall phenotype or be impacted by the loss of CaMKK2 in NK cells. Thus, a model was developed in which CaMKK2 was selectively ablated in NK cells in immune competent animals (FIG.13A). Specifically, mice with floxed CaMKK2 alleles were crossed to mice expressing cre under the control of the Ncr1 (NKp46) promoter, which is only active in NK cells. Experimental and control mice both express one copy of the Ncr1-cre construct, but only the experimental group contain floxed CaMKK2 alleles. Experimental and control mice were injected via tail vein with luciferase- expressing, GFP+B16-F10 tumor cells, and tumor burden was monitored via IVIS. Within two weeks of tumor injection, the group with floxed CaMKK2 alleles exhibited a significantly greater systemic B16-F10 metastatic tumor burden than control mice (FIGS.13B-13C). These studies revealed that ablation of CaMKK2 in the NK cell compartment reduces immune surveillance even in immune competent animals. An extensive immune profiling of post-mortem lung tissue was performed, but no downstream immunologic changes resulting from CaMKK2 ablation in NK cells was identified. As expected, the total number of NK cells was reduced in the CaMKK2fl / flgroup, but the prevalence of B-cells, neutrophils, dendritic cells, T-cell subsets, and macrophage subsets were unaltered (FIG.13D). Thus, the reduced tumor burden associated with CaMKK2 expression in NK cells was likely mediated by direct changes in the size of the effector NK cell population.Docket No.028193-0031-WO01 / 7879 Example 7 CaMKK2 Upregulation is Driven by a Small Molecule Tumor Factor [000172] A mechanism by which the tumor environment influences CaMKK2 expression in NK cells was sought. Many components of the tumor environment could trigger this transcriptional event, including cell-cell interactions or soluble factors. Proteins expressed on tumor cells or other tumor-associated cells may engage specific membrane receptors on NK cells, triggering signaling pathways that alter transcriptional state. For example, when the KLGR1 receptor binds E-cadherin, AMPK dephosphorylation is inhibited, which leads to increased telomerase expression in NK cells. Similarly, soluble factors in the extracellular tumor environment could alter transcription. The two major classes of soluble factors are (1) proteins, which includes cytokines and chemokines, and (2) small molecules, which includes metabolites, hormones, and lipids. A wide range of soluble factors are known to alter the transcriptional state of NK cells, including IL15, TGF-ȕ, and picolinic acid. Previous experiments showed that TCM exposure was sufficient to upregulate CaMKK2 in a pure population of NK cells, negating cell-cell interactions as a likely driver of CaMKK2 expression. Thus, identifying soluble factors sufficient to upregulate CaMKK2 was pursued. [000173] To distinguish between protein and non-protein factors, the effects of normal TCM and heat inactivated TCM on NK cells were compared in vitro. By heating TCM to 50 degrees for 30 minutes, most proteinaceous factors are denatured and lose their normal signaling capacities, but in general small molecules retain their structures and functions. Thus, if TCM loses its capacity to upregulate CaMKK2 when heat-inactivated, then it is implied that a protein factor(s) must be required for upregulation. Interestingly, heat inactivated EO771 TCM produced the same drop in viability as normal TCM when NK cells are treated with the CC3240 LDD (FIGS.14A-14C). These data indicated that small molecule factors are sufficient to mediate CaMKK2 upregulation, and protein factors are uninvolved. [000174] To identify candidate small molecule factors affecting CaMKK2 expression, a metabolomics screen of tumor-conditioned media was conducted. Previous data demonstrated that both EO771 TCM and A7C11 TCM facilitate CaMKK2 upregulation in NK cells ex vivo (FIG. 4G; FIGS.5F-5H). Thus, mass spectrometry was ysed to compare the metabolite composition of these two TCM types against normal media. The Duke Molecular Physiology Institute used liquid chromatography mass spectrometry to identify the relative abundance of 217 small molecule metabolites. A statistical test was then run to prioritize specific metabolites for further investigation, selecting metabolites that were increased inDocket No.028193-0031-WO01 / 7879 both EO771 and A7C11 TCM compared to normal media by a statistically significant factor of two-fold or more (p<0.05). This screen produced 25 candidate factors, which was narrowed to 12 factors by increasing the threshold to an eight-fold increase. These 12 factors included the vitamin derivatives 1-methylnicotinamide and pyridoxal, the catecholamine metabolite dopaminochrome, the amino acid derivatives ketoleucine, 2- oxyglutarate, and N-acetylmethionine, the ribonucleotides UMP and N2-Formyl-N1-(5- phospho-D-ribosyl)glycinamide, and the anaerobic fermentation byproduct lactate. Example 8 Intracellular Lactic Acid Accumulation Drives CaMKK2 Upregulation [000175] The metabolites 1-methylnicotinamide and lactate immediately stood out as both compounds have established mechanistic roles in tumor immunity. 1-methylnicotinamide is known to induce TNFĮ production by tumor-infiltrating T cells, and lactate is known to affect the survival, activity, and functional phenotype of most immune populations in the tumor environment. None of the other candidate factors have known roles in tumor immunology. Given lactate’s multifaceted role in tumor pathobiology, it was elected to evaluate its role in NK cell function before proceeding to a more comprehensive study of the other metabolites identified. Splenic NK cells harvested from CaMKK2 reporter mice were cultured in 50% EO771 TCM, normal media containing 10 mM lactic acid, or normal media alone, and eGFP fluorescence was measured via flow cytometry as a surrogate for CaMKK2 expression. A lactic acid concentration of 10 mM is representative of a typical solid tumor in humans. At 24 hours, there was no change in eGFP expression in the TCM and lactic acid groups (FIG. 15A). However, by 96 hours, both the TCM and lactic acid-treated NK cells showed a significant increase in eGFP expression compared to the normal media control (FIG.15B). A Western immunoblot confirmed these results, showing that exposure to 10 mM lactic acid for 96 hours was sufficient to produce robust CaMKK2 expression, whereas NK cells cultured in normal media show minimal expression (FIG.15C). [000176] Lactic acid and TCM caused similar phenotypes in CaMKK2-deficient NK cells, providing further evidence that lactic acid is the key component of TCM driving increased CaMKK2 expression. CaMKK2 ablation had no effect on NK cells cultured in normal media because the enzyme is minimally expressed under these conditions. In lactic acid-treated NK cells, however, CaMKK2 ablation produced a significant reduction in NK cell viability and proliferation state, identical to the phenotype observed in TCM-treated NK cells (FIGS.15D- 15E). Finally, the fact that the CaMKK2-eGFP reporter model showed the same rapid expression increase as Western blots indicated that transcriptional changes drive CaMKK2Docket No.028193-0031-WO01 / 7879 upregulation, rather than increased post-translational protein stabilization. If protein stabilization were responsible for increased CaMKK2 levels, there would be no parallel increase in the eGFP reporter protein. [000177] The next goal was to elucidate the mechanism by which lactic acid modulates CaMKK2 transcription. Possible mechanisms include signaling through GPR81, a cell- surface G protein-coupled receptor (GPCR) activated by high concentrations of extracellular lactate. According to the Immgen database, however, NK cells do not express detectable levels of GPR81 (FIG.16A). Alternatively, lactate can enter cells and directly modulate transcriptional activity. As discussed earlier, intracellular lactic acid is known to activate the HIF1Į and NRF2 transcription factors, inhibit various HDAC enzymes, and directly modify histone activity through “lactylation” of lysine residues. Indeed, NK cells expressed low-to- moderate levels of the classic lactate shuttles MCT1 and MCT4 (FIGS.16D-16E), suggesting a mechanism by which lactate can be imported into the cells. Through acidification of the tumor environment, lactic acid can indirectly trigger pH sensing GPCRs or cause intracellular acidification, which alters many signaling pathways. Interestingly, NK cells expressed at least two of the four known pH-sensing GPCRs, GPR65 and GPR132 (FIGS.16B-16C), raising the possibility of CaMKK2 regulation through pH-mediated transcriptional effects. [000178] The mechanistic studies began by probing the importance of intracellular lactate import through the MCT1 and MCT4 channels. The monocarboxylate transporter family requires a proton gradient to passively shuttle lactate and pyruvate across the cell membrane. Due to enhanced acidity and proton availability, the MCT family is particularly active in the tumor environment. To test whether these transporters play a role in lactate- mediated CaMKK2 expression, CaMKK2-eGFP reporter NK cells were cultured in 10 mM lactic acid, with or without the MCT1 inhibitor AZD3965. AZD3965 is an MCT1-selective competitive inhibitor currently being explored as a treatment for a variety of cancers. As in prior studies, reporter NK cells treated with lactic acid showed a significant increase in eGFP fluorescence compared to normal media conditions, but in this study, it was demonstrated that treatment with AZD3965 completely blocked this effect (FIGS.17A-17B). These data suggested that MCT1-mediated intracellular import of lactate plays an important role in CaMKK2 upregulation. Interestingly, NK cells treated with sodium lactate exhibited no increase in eGFP expression (FIGS.17A-17B), likely because a proton gradient is required to power MCT import activity. [000179] To confirm these results in a WT model, Western immunoblots were conducted on WT splenic NK cells cultured with lactic acid and MCT inhibitors. Data from the reporterDocket No.028193-0031-WO01 / 7879 model suggested that acidification is not responsible for CaMKK2 upregulation as intracellular import of the lactate molecule is necessary for this response, not exposure to free protons generated by the dissociation of lactic acid. To definitively control for pH effects, however, this experiment used heavily buffered media containing 25 mM HEPES and 25 mM PIPES with a fixed pH of 7.4. Media pH was identical in all samples throughout the experiment, except for one sample whose pH was set to 6.4 (FIG.17C). The immunoblot demonstrated that 10 mM lactic acid is sufficient to drive CaMKK2 protein upregulation in NK cells, but this upregulation is significantly reduced by MCT1 inhibitor AZD3965 and by pan-MCT inhibitor Į-cyano-4-hydroxycinnamic acid (ĮCHC) (FIG.17C). The more complete reduction of CaMKK2 expression in the ĮCHC group compared to the AZD3965 group may reflect the added effects of MCT4 inhibition. Importantly, media acidification alone did not affect CaMKK2 expression levels (FIG.17C), and acidified media does not replicate the effects of TCM on the survival of CaMKK2-deficient NK cells (FIG. 17D). These data ruled out a specific role for acidification in the regulation of CaMKK2 expression while confirming the necessity of intracellular lactate import. Example 9 Tying CaMKK2 to Human NK Cell Biology [000180] Human and murine NK cells are quite similar, with analogous developmental patterns, effector mechanisms, and functional markers. Thus, the findings in mice are expected to translate to human biology. Interestingly, it was observed that the human NK cell lymphoma line NK92 constitutively expresses robust levels of CaMKK2 (FIG.18A), confirming that human NK cell derivatives are capable of CaMKK2 expression. However, ligand-directed degradation of CaMKK2 with CC3240 had no effect on the survival, proliferation, or activation of NK92 cells, even under stressful TCM conditioning (FIG.18B). CC3240 degraded about 75% of the CaMKK2 protein in NK92 cells (FIG.18C), which is like the reduction in primary mouse NK cells. [000181] Through clinical collaborations on projects unrelated to CaMKK2, single cell RNA sequencing data for tumor-infiltrating human immune cells was acquired. Through UMAP dimension reduction techniques, a diverse repertoire of immune cells recovered from three primary human breast tumors, including NK cell populations, were plotted and analyzed. The NK cell cluster was identified by high expression of Ncr1 (NKp46) mRNA, (FIG.18D), and this cluster was then analyzed for evidence of CaMKK2 expression. The study was valid as CaMKK2 mRNA was observed in the macrophage cluster, but very little CaMKK2 was detected in the tumor-infiltrating NK cells (FIG.18E). However, this study had severalDocket No.028193-0031-WO01 / 7879 limitations, including the small sample size, limited NK cell infiltration, relatively low sequencing penetrance, and unknown lactic acid concentrations within the tumor environment. As discussed earlier, NK cell infiltration in primary tumors is generally quite poor, and the small number of NK cells recovered from these three specific samples may not have penetrated the tumor capsule or been exposed to interior regions with pronounced lactic acid accumulation. Moreover, CaMKK2 detection in the macrophage cluster was rather sparse, suggesting that the expression threshold for detection was quite high in this study. Example 10 Effects of CaMKK2 Ablation on Peripheral NK Cells [000182] Most research has focused on tumor-infiltrating NK cells, not peripheral NK cells in tumor-bearing animals. This is largely due to early experiments in which tumor-bearing CaMKK2-eGFP reporter mice failed to show upregulation of CaMKK2 in splenic NK cells. However, in two of the metastasis studies, NK cells from spleens and tumors were recovered, and, surprisingly, a significant reduction in total NK cell numbers in the spleens of the CaMKK2-deficient group was observed (FIGS.19A-19B). In these studies, WT and KO splenic NK cells exhibited comparable levels of viability (FIGS.19A-19B), implying that variations in total cell number are not driven by differences in survival. Differences in proliferation rate may explain these results. Example 11 Discussion [000183] FIG.1 schematically shows key metabolic regulatory pathways in natural killer (NK) cells. CaMKK2 has been studied extensively in the context of tumor cells and myeloid immune cells, but this enzyme was not previously known to play a role in NK cell immunity. The studies described herein demonstrated that a subset of CD11b+NK cells express CaMKK2 at baseline, though this subset lacks a specific identity as CaMKK2 expression does not closely correlate with common NK cell functional markers. CD11b+NK cells demonstrated a binary pattern of CaMKK2 expression as reporter cells are either completely negative or distinctly positive for eGFP. Thus, the mechanism(s) regulating CaMKK2 levels may involve a “threshold effect” whereby exposure to a specific level of a stimulating factor increases transcription, causing some cells to stochastically express the enzyme while others do not. Upon exposure to tumor conditions, CaMKK2 is robustly expressed in all subsets of NK cells, including immature CD11bíNK cells and NKT cells. Thus, a regulatoryDocket No.028193-0031-WO01 / 7879 apparatus is likely conserved across the extended NK cell family, enabling CaMKK2 transcription when the proper stimulus is detected. CD11b+NK cells may simply be more sensitive to this stimulus, explaining the mixed baseline expression. Increased CaMKK2 expression is likely due to transcriptional upregulation rather than protein stabilization because the eGFP reporter model shows the same increase as Western blots. Finally, CaMKK2 expression was not observed in any post-thymic T-cell subset, indicating that CaMKK2 modulation is specific to the NK cell lineage as other closely related lymphoid populations never express this enzyme. [000184] In vitro functional assays demonstrated that CaMKK2 expression improves the general fitness of tumor-conditioned NK cells through a novel scaffold mechanism. Ablation of CaMKK2 through genetic means or ligand-directed degradation increases apoptosis and reduces viability in tumor-conditioned NK cells. CaMKK2 ablation also causes cell cycle quiescence, restricts proliferation, and reduces chemotaxis under tumor conditions. However, CaMKK2 ablation does not affect the cytotoxic potency of individual tumor- conditioned NK cells. Considering the mitochondria’s multifaceted role in regulating apoptosis, cell cycle progression, and even chemotaxis, it was hypothesized that CaMKK2 could be influencing NK cell fitness through mitochondrial regulation. However, CaMKK2 ablation has no effect on mitochondria mass, membrane potential, or reactive oxygen species, which suggests a non-mitochondrial mechanism by which CaMKK2 influences NK cell biology. In contrast to genetic ablation or ligand-directed degradation of CaMKK2, competitive inhibition of this enzyme has no effect on tumor-conditioned NK cells, indicating that CaMKK2 serves a unique scaffold function independent of its kinase activity. The nature and implications of this scaffold function are discussed in greater detail later in this section. [000185] Metastasis studies consistently show increased metastatic progression in mice with CaMKK2-deficient NK cells, establishing an important role for CaMKK2 in NK cell tumor immunity. These results were repeated using three models of NK cell-intrinsic CaMKK2 ablation and two different NK cell-sensitive tumor models, B16-F10 and EO771ȕ2mí / í. Tumor cells recovered from the lungs of mice with CaMKK2-deficient NK cells showed significantly lower MHC I expression than WT controls, an immunoediting phenotype consistent with reduced NK cell anti-tumor activity. Moreover, immune profiling showed that total NK cell counts were significantly lower in the lungs of the CaMKK2-deficient groups despite their larger tumor burdens, and the NK cells that were present showed reduced viability and proliferative capacity. This in vivo immune profiling data is consistent with the in vitro data generated using tumor-conditioned media. Even in fully immune competent mice withDocket No.028193-0031-WO01 / 7879 several intact mechanisms of anti-tumor immunity, NK cell-specific ablation of CaMKK2 led to increased metastatic burden. Extensive immune profiling of lung tissue from this study failed to show changes in the infiltration or polarization of any immune populations besides NK cells, highlighting (1) the importance of effector NK cell activity in controlling certain tumor types and (2) the importance of CaMKK2 in preserving NK cell function. [000186] Based on the data presented thus far, CaMKK2 expression reduces metastatic progression by improving the fitness of NK cells within metastatic nodules, not by increasing NK cell recruitment to tumors or by enhancing NK cell anti-tumor surveillance in circulation. CaMKK2 is only expressed at appreciable levels in NK cells exposed to tumor conditions for at least 72 hours, and peripheral NK cells isolated from tumor-bearing mice showed no evidence of CaMKK2 upregulation in the eGFP reporter model. Thus, NK cells in circulation likely not express CaMKK2, and WT and CaMKK2 KO NK cells in circulation likely not display any phenotypic differences, including their ability to migrate to the tumor or mediate cytotoxic activity in circulation. Indeed, three of the four metastasis studies showed a growing divergence between the WT and KO groups as the metastatic tumors grew larger, with two of the studies developing differences in metastatic burden only at the latest timepoint (FIG.10B; FIG.12C). These results are consistent with NK cells being conditioned by an established tumor environment before CaMKK2 is upregulated and begins to play an important role in NK immunity. Moreover, in the EO771ȕ2mí / ímodel, NK cells were intravenously injected 48 hours after the tumor cells, meaning that many tumor cells had already extravasated and begun colonizing tissues by the time NK cells were introduced into the system. Thus, NK cell activity against circulating EO771 cells is unlikely to explain the large differences in metastatic progression between the WT and CaMKK2-deficient groups. [000187] Although NK cell recruitment from the periphery to tumor nodules is unlikely to explain the metastasis phenotype, impaired intra-tumoral migration may contribute to increased metastatic growth in the CaMKK2-deficient groups. The in vitro migration assay showed that CaMKK2 ablation impairs baseline migration and CCL4 chemotaxis in tumor- conditioned NK cells. These findings could translate to reduced infiltration of CaMKK2- deficient NK cells into deep, less-vascularized portions of a tumor nodule. To test this hypothesis in vivo, immunohistochemistry staining was conducted on large lung nodules recovered from mice with WT or CaMKK2-deficient NK cells, looking for reduced “deep tumor” infiltration in the CaMKK2-deficient group. A reasonable explanation for differences in metastatic progression in the WT versus CaMKK2-deficient mice may involve the survival and proliferation of NK cells within the local tumor environment.Docket No.028193-0031-WO01 / 7879 Example 12 Inducing Expression of CaMKK2 in Isolated NK Cells [000188] Peripheral blood mononuclear cells (PBMCs) will be separated from whole patient blood through leukapheresis. Anti-CD3 immunomagnetic particles will be used to deplete T-cells, and the remaining PBMCs will be co-cultured for 2 weeks with irradiated IL- 15 expressing K562 to expand the NK cell fraction. Using a viral vector, the expanded population of NK cells will be transduced with a retroviral genetic construct promoting constitutive CaMKK2 expression, and the modified NK cells will be autologously transplanted back into the original patient / donor. *** [000189] The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. [000190] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents. [000191] All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document were individually indicated to be incorporated by reference for all purposes. [000192] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses: [000193] Clause 1. A method of increasing survival and / or proliferation of a natural killer (NK) cell in a tumor of a subject, the method comprising: isolating the natural killer (NK) cell from a biological sample from the subject to generate an isolated NK cell; administering toDocket No.028193-0031-WO01 / 7879 the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell; and administering to the subject the modified NK cell. [000194] Clause 2. A method of increasing the level of CaMKK2 in a natural killer (NK) cell in a tumor of a subject, the method comprising: isolating the natural killer (NK) cell from a biological sample from the subject to generate an isolated NK cell; administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell; and administering to the subject the modified NK cell. [000195] Clause 3. A method of treating cancer in a subject, the method comprising: isolating a natural killer (NK) cell from a biological sample from the subject to generate an isolated NK cell; administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell; and administering to the subject the modified NK cell. [000196] Clause 4. The method of any one of clauses 1-3, wherein the modified NK cell has reduced rate of apoptosis compared to a control NK cell. [000197] Clause 5. The method of any one of clauses 1-4, wherein the modified NK cell has an increased rate of replication and / or growth compared to a control NK cell. [000198] Clause 6. The method of any one of clauses 1-5, wherein the CaMKK2 expressed in the modified NK cell maintains scaffolding activity. [000199] Clause 7. The method of any one of clauses 1-6, wherein the modified NK cell constitutively expresses a level of CaMKK2 greater than that of a control NK cell. [000200] Clause 8. The method of any one of clauses 1-7, wherein the modified NK cell expresses a level of CaMKK2 that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8- fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold greater than that of a control NK cell. [000201] Clause 9. The method of any one of clauses 1-8, wherein the administration of the modified NK cell decreases the rate of progression of tumor growth in the subject, as compared to the rate of progression of tumor growth prior to the modified NK cell treatment.Docket No.028193-0031-WO01 / 7879 [000202] Clause 10. The method of any one of clauses 1-9, wherein the administration of the modified NK cell decreases the rate of cancer metastasis in the subject, as compared to the rate of cancer metastasis prior to the modified NK cell treatment. [000203] Clause 11. The method of any one of clauses 1-10, wherein the administration of the modified NK cell decreases the size of a tumor in the subject, as compared to the size of the tumor prior to the modified NK cell treatment. [000204] Clause 12. The method of any one of clauses 1-11, wherein the method further comprises administering to the isolated NK cell a polynucleotide encoding LDH to increase the level of CaMKK2 in the isolated NK cell. [000205] Clause 13. The method of clause 12, wherein the LDH comprises a polypeptide having the amino acid sequence of SEQ ID NO: 4, and / or wherein the polynucleotide encoding LDH comprises the sequence of SEQ ID NO: 5 or 6. [000206] Clause 14. The method of any one of clauses 1-13, wherein a vector comprises the polynucleotide encoding CaMKK2. [000207] Clause 15. The method of clause 14, wherein the vector is an AAV vector. [000208] Clause 16. The method of any one of clauses 1-15, wherein the polynucleotide encoding CaMKK2 comprises the sequence of SEQ ID NO: 2 or 3, and / or wherein the CaMKK2 comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1. [000209] Clause 17. The method of any one of clauses 1-16, wherein the modified NK cell is administered to the subject once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months. [000210] Clause 18. The method of any one of clauses 1-16, wherein the modified NK cell is administered to the subject for 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years. [000211] Clause 19. The method of any one of clauses 1-18, wherein the method further comprises administering to the subject at least one additional therapy.Docket No.028193-0031-WO01 / 7879 [000212] Clause 20. The method of clause 19, wherein the at least one additional therapy is selected from chemotherapy, immunotherapy, radiation therapy, hormone therapy, targeted drug therapy, cryoablation, and surgery, or a combination thereof. [000213] Clause 21. The method of clause 20, wherein the chemotherapy is selected from an antimitotic agent, an alkylating agent, an antimetabolite, an antimicrotubule agent, a topoisomerase inhibitor, a cytotoxic agent, a cell cycle inhibitor, a growth factor inhibitor, a histone deacetylase (HDAC) inhibitor, and an inhibitor of a pathway that cross-talks with and activates ER transcriptional activity, or a combination thereof. [000214] Clause 22. The method of any one of clauses 1-21, wherein the subject has a solid cancer. [000215] Clause 23. The method of clause 22, wherein the solid cancer comprises melanoma, colon cancer, breast cancer, lung cancer, head and neck cancer, laryngeal cancer, oral cancer, esophageal cancer, gastric cancer, stomach cancer, colon cancer, bladder cancer, liver cancer, pancreatic cancer, kidney cancer, brain cancer, bone cancer, glioblastoma, sarcoma, ovarian cancer, endometrial cancer, or prostate cancer, or a combination thereof. [000216] Clause 24. A composition for increasing survival and / or proliferation of a natural killer (NK) cell, the composition comprising a polynucleotide encoding CaMKK2 to express a level of CaMKK2 in the NK cell greater than that of a control NK cell. [000217] Clause 25. The method of clause 24, wherein the polynucleotide encoding CaMKK2 comprises the sequence of SEQ ID NO: 4 or 5. SEQUENCES SEQ ID NO: 1 Polypeptide sequence of human CaMKK2 MSSCVSSQPSSNRAAPQDELGGRGSSSSESQKPCEALRGLSSLSIHLGMESFIVVTECEPGC AVDLGLARDRPLEADGQEVPLDSSGSQARPHLSGRKLSLQERSQGGLAAGGSLDMNGRCICP SLPYSPVSSPQSSPRLPRRPTVESHHVSITGMQDCVQLNQYTLKDEIGKGSYGVVKLAYNEN DNTYYAMKVLSKKKLIRQAGFPRRPPPRGTRPAPGGCIQPRGPIEQVYQEIAILKKLDHPNV VKLVEVLDDPNEDHLYMVFELVNQGPVMEVPTLKPLSEDQARFYFQDLIKGIEYLHYQKIIH RDIKPSNLLVGEDGHIKIADFGVSNEFKGSDALLSNTVGTPAFMAPESLSETRKIFSGKALD VWAMGVTLYCFVFGQCPFMDERIMCLHSKIKSQALEFPDQPDIAEDLKDLITRMLDKNPESR IVVPEIKLHPWVTRHGAEPLPSEDENCTLVEVTEEEVENSVKHIPSLATVILVKTMIRKRSF GNPFEGSRREERSLSAPGNLLTKQGSEDNLQGTDPPPVGEEEVLLLDocket No.028193-0031-WO01 / 7879 SEQ ID NO: 2 DNA sequence for human CaMKK2 atgtcatcatgtgtctctagccagcccagcagcaaccgggccgccccccaggatgagctggg gggcaggggcagcagcagcagcgaaagccagaagccctgtgaggccctgcggggcctctcat ccttgagcatccacctgggcatggagtccttcattgtggtcaccgagtgtgagccgggctgt gctgtggacctcggcttggcgcgggaccggcccctggaggccgatggccaagaggtccccct tgactcctccgggtcccaggcccggccccacctctccggtcgcaagctgtctctgcaagagc ggtcccagggtgggctggcagccggtggcagcctggacatgaacggacgctgcatctgcccg tccctgccctactcacccgtcagctccccgcagtcctcgcctcggctgccccggcggccgac agtggagtctcaccacgtctccatcacgggtatgcaggactgtgtgcagctgaatcagtata ccctgaaggatgaaattggaaagggctcctatggtgtcgtcaagttggcctacaatgaaaat gacaatacctactatgcaatgaaggtgctgtccaaaaagaagctgatccggcaggccggctt tccacgtcgccctccaccccgaggcacccggccagctcctggaggctgcatccagcccaggg gccccattgagcaggtgtaccaggaaattgccatcctcaagaagctggaccaccccaatgtg gtgaagctggtggaggtcctggatgaccccaatgaggaccatctgtacatggtgttcgaact ggtcaaccaagggcccgtgatggaagtgcccaccctcaaaccactctctgaagaccaggccc gtttctacttccaggatctgatcaaaggcatcgagtacttacactaccagaagatcatccac cgtgacatcaaaccttccaacctcctggtcggagaagatgggcacatcaagatcgctgactt tggtgtgagcaatgaattcaagggcagtgacgcgctcctctccaacaccgtgggcacgcccg ccttcatggcacccgagtcgctctctgagacccgcaagatcttctctgggaaggccttggat gtttgggccatgggtgtgacactatactgctttgtctttggccagtgcccattcatggacga gcggatcatgtgtttacacagtaagatcaagagtcaggccctggaatttccagaccagcccg acatagctgaggacttgaaggacctgatcacccgtatgctggacaagaaccccgagtcgagg atcgtggtgccggaaatcaagctgcacccctgggtcacgaggcatggggcggagccgttgcc gtcggaggatgagaactgcacgctggtcgaagtgactgaagaggaggtcgagaactcagtca aacacattcccagcttggcaaccgtgatcctggtgaagaccatgatacgtaaacgctccttt gggaacccattcgagggcagccggcgggaggaacgctcactgtcagcgcctggaaacttgct cacgaagcaaggcagcgaagacaacctccagggcaccgacccgccccccgtgggggaggagg aagtgctcttgttg SEQ ID NO: 3 RNA sequence for human CaMKK2 augucaucaugugucucuagccagcccagcagcaaccgggccgccccccaggaugagcuggg gggcaggggcagcagcagcagcgaaagccagaagcccugugaggcccugcggggccucucau ccuugagcauccaccugggcauggaguccuucauuguggucaccgagugugagccgggcugu gcuguggaccucggcuuggcgcgggaccggccccuggaggccgauggccaagaggucccccu ugacuccuccgggucccaggcccggccccaccucuccggucgcaagcugucucugcaagagc ggucccagggugggcuggcagccgguggcagccuggacaugaacggacgcugcaucugcccg ucccugcccuacucacccgucagcuccccgcaguccucgccucggcugccccggcggccgac aguggagucucaccacgucuccaucacggguaugcaggacugugugcagcugaaucaguaua cccugaaggaugaaauuggaaagggcuccuauggugucgucaaguuggccuacaaugaaaau gacaauaccuacuaugcaaugaaggugcuguccaaaaagaagcugauccggcaggccggcuu uccacgucgcccuccaccccgaggcacccggccagcuccuggaggcugcauccagcccaggg gccccauugagcagguguaccaggaaauugccauccucaagaagcuggaccaccccaaugug gugaagcugguggagguccuggaugaccccaaugaggaccaucuguacaugguguucgaacu ggucaaccaagggcccgugauggaagugcccacccucaaaccacucucugaagaccaggccc guuucuacuuccaggaucugaucaaaggcaucgaguacuuacacuaccagaagaucauccac cgugacaucaaaccuuccaaccuccuggucggagaagaugggcacaucaagaucgcugacuu uggugugagcaaugaauucaagggcagugacgcgcuccucuccaacaccgugggcacgcccg ccuucauggcacccgagucgcucucugagacccgcaagaucuucucugggaaggccuuggau guuugggccaugggugugacacuauacugcuuugucuuuggccagugcccauucauggacgaDocket No.028193-0031-WO01 / 7879 gcggaucauguguuuacacaguaagaucaagagucaggcccuggaauuuccagaccagcccg acauagcugaggacuugaaggaccugaucacccguaugcuggacaagaaccccgagucgagg aucguggugccggaaaucaagcugcaccccugggucacgaggcauggggcggagccguugcc gucggaggaugagaacugcacgcuggucgaagugacugaagaggaggucgagaacucaguca aacacauucccagcuuggcaaccgugauccuggugaagaccaugauacguaaacgcuccuuu gggaacccauucgagggcagccggcgggaggaacgcucacugucagcgccuggaaacuugcu cacgaagcaaggcagcgaagacaaccuccagggcaccgacccgccccccgugggggaggagg aagugcucuuguug SEQ ID NO: 4 Polypeptide sequence of human LDH MATLKDQLIYNLLKEEQTPQNKITVVGVGAVGMACAISILMKDLADELALVDVIEDKLKG EMMDLQHGSLFLRTPKIVSGKDYNVTANSKLVIITAGARQQEGESRLNLVQRNVNIFKFI IPNVVKYSPNCKLLIVSNPVDILTYVAWKISGFPKNRVIGSGCNLDSARFRYLMGERLGV HPLSCHGWVLGEHGDSSVPVWSGMNVAGVSLKTLHPDLGTDKDKEQWKEVHKQVVESAYE VIKLKGYTSWAIGLSVADLAESIMKNLRRVHPVSTMIKGLYGIKDDVFLSVPCILGQNGI SDLVKVTLTSEEEARLKKSADTLWGIQKELQF SEQ ID NO: 5 DNA sequence for human LDH atggcaactctaaaggatcagctgatttataatcttctaaaggaagaacagaccccccagaa taagattacagttgttggggttggtgctgttggcatggcctgtgccatcagtatcttaatga aggacttggcagatgaacttgctcttgttgatgtcatcgaagacaaattgaagggagagatg atggatctccaacatggcagccttttccttagaacaccaaagattgtctctggcaaagacta taatgtaactgcaaactccaagctggtcattatcacggctggggcacgtcagcaagagggag aaagccgtcttaatttggtccagcgtaacgtgaacatatttaaattcatcattcctaatgtt gtaaaatacagcccgaactgcaagttgcttattgtttcaaatccagtggatatcttgaccta cgtggcttggaagataagtggttttcccaaaaaccgtgttattggaagtggttgcaatctgg attcagcccgattccgttacctgatgggggaaaggctgggagttcacccattaagctgtcat gggtgggtccttggggaacatggagattccagtgtgcctgtatggagtggaatgaatgttgc tggtgtctctctgaagactctgcacccagatttagggactgataaagataaggaacagtgga aagaggttcacaagcaggtggttgagagtgcttatgaggtgatcaaactcaaaggctacaca tcctgggctattggactctctgtagcagatttggcagagagtataatgaagaatcttaggcg ggtgcacccagtttccaccatgattaagggtctttacggaataaaggatgatgtcttcctta gtgttccttgcattttgggacagaatggaatctcagaccttgtgaaggtgactctgacttct gaggaagaggcccgtttgaagaagagtgcagatacactttgggggatccaaaaggagctgca attt SEQ ID NO: 6 RNA sequence for human LDH auggcaacucuaaaggaucagcugauuuauaaucuucuaaaggaagaacagaccccccagaa uaagauuacaguuguugggguuggugcuguuggcauggccugugccaucaguaucuuaauga aggacuuggcagaugaacuugcucuuguugaugucaucgaagacaaauugaagggagagaug auggaucuccaacauggcagccuuuuccuuagaacaccaaagauugucucuggcaaagacua uaauguaacugcaaacuccaagcuggucauuaucacggcuggggcacgucagcaagagggag aaagccgucuuaauuugguccagcguaacgugaacauauuuaaauucaucauuccuaauguu guaaaauacagcccgaacugcaaguugcuuauuguuucaaauccaguggauaucuugaccua cguggcuuggaagauaagugguuuucccaaaaaccguguuauuggaagugguugcaaucugg auucagcccgauuccguuaccugaugggggaaaggcugggaguucacccauuaagcugucau ggguggguccuuggggaacauggagauuccagugugccuguauggaguggaaugaauguugc uggugucucucugaagacucugcacccagauuuagggacugauaaagauaaggaacaguggaDocket No.028193-0031-WO01 / 7879 aagagguucacaagcaggugguugagagugcuuaugaggugaucaaacucaaaggcuacaca uccugggcuauuggacucucuguagcagauuuggcagagaguauaaugaagaaucuuaggcg ggugcacccaguuuccaccaugauuaagggucuuuacggaauaaaggaugaugucuuccuua guguuccuugcauuuugggacagaauggaaucucagaccuugugaaggugacucugacuucu gaggaagaggcccguuugaagaagagugcagauacacuuugggggauccaaaaggagcugca auuu B2M-1F: 5’-CACCGAGTATACTCACGCCACCCAC-3’ (SEQ ID NO: 7) B2M-1R: 5’-AAACGTGGGTGGCGTGAGTATACTC-3’ (SEQ ID NO: 8) B2M-2F: 5’-CACCGAGTCGTCAGCATGGCTCGCT-3’ (SEQ ID NO: 9) B2M-2R: 5’-AAACAGCGAGCCATGCTGACGACTC-3’ (SEQ ID NO: 10)

Claims

Docket No.028193-0031-WO01 / 7879 CLAIMS 1. A method of increasing survival and / or proliferation of a natural killer (NK) cell in a tumor of a subject, the method comprising: isolating the natural killer (NK) cell from a biological sample from the subject to generate an isolated NK cell; administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell; and administering to the subject the modified NK cell.

2. A method of increasing the level of CaMKK2 in a natural killer (NK) cell in a tumor of a subject, the method comprising: isolating the natural killer (NK) cell from a biological sample from the subject to generate an isolated NK cell; administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell; and administering to the subject the modified NK cell.

3. A method of treating cancer in a subject, the method comprising: isolating a natural killer (NK) cell from a biological sample from the subject to generate an isolated NK cell; administering to the isolated NK cell a polynucleotide encoding CaMKK2 to generate a modified NK cell, wherein the modified NK cell expresses a level of CaMKK2 greater than that of a control NK cell; and administering to the subject the modified NK cell.

4. The method of any one of claims 1-3, wherein the modified NK cell has reduced rate of apoptosis compared to a control NK cell.

5. The method of any one of claims 1-4, wherein the modified NK cell has an increased rate of replication and / or growth compared to a control NK cell.

6. The method of any one of claims 1-5, wherein the CaMKK2 expressed in the modified NK cell maintains scaffolding activity.Docket No.028193-0031-WO01 / 7879 7. The method of any one of claims 1-6, wherein the modified NK cell constitutively expresses a level of CaMKK2 greater than that of a control NK cell.

8. The method of any one of claims 1-7, wherein the modified NK cell expresses a level of CaMKK2 that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9- fold, 9.5-fold, or 10-fold greater than that of a control NK cell.

9. The method of any one of claims 1-8, wherein the administration of the modified NK cell decreases the rate of progression of tumor growth in the subject, as compared to the rate of progression of tumor growth prior to the modified NK cell treatment.

10. The method of any one of claims 1-9, wherein the administration of the modified NK cell decreases the rate of cancer metastasis in the subject, as compared to the rate of cancer metastasis prior to the modified NK cell treatment.

11. The method of any one of claims 1-10, wherein the administration of the modified NK cell decreases the size of a tumor in the subject, as compared to the size of the tumor prior to the modified NK cell treatment.

12. The method of any one of claims 1-11, wherein the method further comprises administering to the isolated NK cell a polynucleotide encoding LDH to increase the level of CaMKK2 in the isolated NK cell.

13. The method of claim 12, wherein the LDH comprises a polypeptide having the amino acid sequence of SEQ ID NO: 4, and / or wherein the polynucleotide encoding LDH comprises the sequence of SEQ ID NO: 5 or 6.

14. The method of any one of claims 1-13, wherein a vector comprises the polynucleotide encoding CaMKK2.

15. The method of claim 14, wherein the vector is an AAV vector.

16. The method of any one of claims 1-15, wherein the polynucleotide encoding CaMKK2 comprises the sequence of SEQ ID NO: 2 or 3, and / or wherein the CaMKK2 comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1.Docket No.028193-0031-WO01 / 7879 17. The method of any one of claims 1-16, wherein the modified NK cell is administered to the subject once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months.

18. The method of any one of claims 1-16, wherein the modified NK cell is administered to the subject for 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years.

19. The method of any one of claims 1-18, wherein the method further comprises administering to the subject at least one additional therapy.

20. The method of claim 19, wherein the at least one additional therapy is selected from chemotherapy, immunotherapy, radiation therapy, hormone therapy, targeted drug therapy, cryoablation, and surgery, or a combination thereof.

21. The method of claim 20, wherein the chemotherapy is selected from an antimitotic agent, an alkylating agent, an antimetabolite, an antimicrotubule agent, a topoisomerase inhibitor, a cytotoxic agent, a cell cycle inhibitor, a growth factor inhibitor, a histone deacetylase (HDAC) inhibitor, and an inhibitor of a pathway that cross-talks with and activates ER transcriptional activity, or a combination thereof.

22. The method of any one of claims 1-21, wherein the subject has a solid cancer.

23. The method of claim 22, wherein the solid cancer comprises melanoma, colon cancer, breast cancer, lung cancer, head and neck cancer, laryngeal cancer, oral cancer, esophageal cancer, gastric cancer, stomach cancer, colon cancer, bladder cancer, liver cancer, pancreatic cancer, kidney cancer, brain cancer, bone cancer, glioblastoma, sarcoma, ovarian cancer, endometrial cancer, or prostate cancer, or a combination thereof.

24. A composition for increasing survival and / or proliferation of a natural killer (NK) cell, the composition comprising a polynucleotide encoding CaMKK2 to express a level of CaMKK2 in the NK cell greater than that of a control NK cell.Docket No.028193-0031-WO01 / 7879 25. The method of claim 24, wherein the polynucleotide encoding CaMKK2 comprises the sequence of SEQ ID NO: 4 or 5.

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