Potentiation of cancer immunotherapy via combination with polyamine blocking therapy

Abstract

Disclosed herein are methods of treating cancer that involve administering polyamine transport inhibitors in conjunction with an immune checkpoint inhibitor for potentiating effects in treating cancers. In certain embodiments polyamine blocking therapy (DFMO + polyamine transport inhibitor administration) in conjunction with checkpoint inhibitor administration is disclosed for treating cancer.

Description

[0001] ATTORNEY DOCKET NO.10669-426PC0 POTENTIATION OF CANCER IMMUNOTHERAPY VIA COMBINATION WITH POLYAMINE BLOCKING THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority to U.S Provisional Application No. 63 / 680,285 filed August 7, 2024, and U.S. Provisional Application No. 63 / 842,202 filed July 11, 2025. The teachings of said applications are incorporated herein, by reference, in their entirety. BACKGROUND Cancer is among the leading causes of death worldwide, accounting for an estimated 8.2 million deaths in 2012 (Globocan 2012, International Agency for Research on Cancer (IARC), World Health Organization, IARC website). The mainstays of treatment for many types of cancer remain conventional chemotherapeutic agents and radiotherapy, which act on dividing cells in a relatively non-specific manner. Most therapeutic agents that have been approved in recent years for treatment of cancer confer only modest increases in average life expectancy and / or have relatively narrow indication(s). There is significant unmet need for innovative approaches for treating cancer. There is a particular need for improved treatment for patients with advanced cancer. BRIEF DESCRIPTION OF DRAWINGS Figure 1. Polyamine metabolism example in pancreatic ductal adenocarcinoma (PDAC) with nodes for DFMO+PTI, i.e., polyamine blocking therapy (PBT) intervention. Abbreviations: AdoMetDC: S-adenosylmethionine decarboxylase; AMXT1501: a Nε-palmitoyl-lysine-spermine conjugate, Hexadecanoic acid (5-amino-5-(3-[4-(3-amino-propylamino)-butylamino]-propylcarbamoyl)-pentyl)- amide; APAO: polyamine oxidase; Arg: arginine; ARG: arginase 1; ATP13A3: an ATPase involved in polyamine transport; AZ: antizyme; AZIN: antizyme inhibitor 1; Cav-1: caveolin-1; CDAP: N- cyclohexyl-1,3-diaminopropane a SMS inhibitor; c-Myc: a transcriptional activator of ODC; dcAdoMet: decarboxylated S-adenosylmethionine; DFMO: difluoromethylornithine; DHPS: deoxyhypusine synthase; DOHH: deoxyhypusine hydroxylase; eIF-5A: eukaryotic initiation factor 5A; GW5074: 3- (3,5-Dibromo-4-hydroxy-benzylidene)-5-iodo-1,3-dihydro-indol-2-one; lasso PTI: N1-(4- [1,4,7]Triazonan-1-yl-butyl)-butane-1,4-diamine; MAT: Methionine adenosyl transferase; ODC: ornithine decarboxylase; SSAT1: spermidine / spermine acetyl transferase; SMOX: spermine oxidase; ATTORNEY DOCKET NO.10669-426PC0 SMS: spermine synthase; SRM: spermidine synthase; trimer44NMe: N-(3,5-Bis-([4-(4-methylamino- butylamino)-butylamino]-methyl)-benzyl)-N'-(4-methylamino-butyl)-butane-1,4-diamine. Figure 2A. Breast cancer response and melanoma responses to DFMO, trimer44NMe PTI and PD-1 antibody (Immune Checkpoint Blockade, ICB). Top scheme outlines experiments evaluating PBT sensitization of resistant tumors to ICB. For panel A, Balb / c mice were orthotopically injected in the mammary fat pad with 4T1 mammary carcinoma cells. For panel C, C57Bl / 6 mice were injected in the dorsal skin with B16F10 melanoma cells. On days 8, 11, and 14, mice were administered ICB (100 µg anti-PD-1 mAb or 100 µg control isotype mAb). On day 16, daily treatment was initiated with either saline or PBT. Panel A) Mean tumor volumes in the 4T1 expt. B) IFN-γ ELISpot and Pentamer analysis showing PBT increases frequency of SIINFEKL-specific CD8+T cells in mice with B16F10 tumors expressing SIINFEKL. C) survival of B16F10 tumor bearing mice. Figure 2D. Using the experimental scheme outlined in the top panel, C57Bl / 6 mice were orthotopically injected with EO771.LMB mammary carcinoma cells (1 x 105cells), then administered ICB (100 µg anti-PD-1 mAb or 100 µg control isotype mAb) on days 10, 13, and 16. On day 12, daily treatment was initiated with either saline or PBT (0.5% DFMO (w / v) in the drinking water plus Trimer44NMe PTI [3 mg / kg, i.p., daily]). Tumor growth was measured with calipers until the mice were sacrificed 5 weeks after tumor cell injection. Mean tumor volume ± SEM. Treatment with ICB ± PBT was discontinued for 3 mice whose tumors had completely regressed at 5 weeks after tumor injection. Three weeks later, these tumor-free mice were re-challenged with the same tumor cells but in an opposite mammary fat pad and observed for tumor growth but with no further treatment. There was no secondary tumor development over the next 2 months. Figure 3. Pancreatic cancer response to DFMO, trimer44NMe PTI and PD-1 antibody. Panel A: Log of total flux (Radiance). Log used since flux values exist in a large range (10*4-10*9) so this keeps the PD1 and PBT bars from becoming indiscernible. Panel B: % of the original tumor bioluminescence (BLI). Tumor total flux recorded at indicated days was divided by total flux of the same tumor on first day of treatment (D0). (B) Quantification of IVIS imaging over the course of treatment (n = 3-4 mice per group). Data are presented as mean ± standard deviation. Comparisons between groups were assessed using two-way ANOVA with Tukey’s multiple comparison. * P<0.05, ** P<0.01, *** P<0.001, and **** P<0.0001. Notes: Combination experienced more animals with tumor loss by day 5, and had less recovery growth even in the combination animal that still had a tumor. This evidences that ATTORNEY DOCKET NO.10669-426PC0 PBT appears to be successful at fighting off regression relative to PD-1 alone. PBT was conducted with 5 days on and weekends off. Panel C: PBT+PD-1 Reduces PDAC Tumor Growth and Significantly Increases Tumor Regression. KPC482GL-2 cells were orthotopically injected into the pancreas of TP53floxed mice (6-8 weeks of age). 12 days post-implantation mice were treated with either vehicle, PBT (0.25% DFMO in drinking water, 4 mg / kg PTI daily injection), 150 µg α-PD-1, or the combination of the two therapies for 17 days. IVIS Imaging at indicated days of individual mice in each treatment group. Bioluminescence is represented as radiance and measured for total flux. Figure 4. Combinational α-PD-1 and polyamine blockade therapy is significantly better than α- PD-1 alone at suppressing pancreatic tumor growth over time. (A) A schematic illustrates the treatment study design. Duplicate arms (arm 1 and arm 2) of the study were combined with C57Bl / 6J mice injected orthotopically into the mouse pancreas with KPC-GFP-Luc2 pancreatic tumor cells. Two weeks later, mice were treated with PBT (0.25% DFMO in the drinking water + 4 mg / kg trimer44NMe PTI injected intraperitoneally daily), with anti-PD-1 (α-PD1, ) antibody (150 μg administered intraperitoneally every 2 days for 3 total doses) and / or in combination. (B) Tumor Luc2 bioluminescence (BLI) of all mice in the duplicate arms was measured over 28 days. BLI is represented as a fold change from BLI at D0. BLI=bioluminescence imaging. Data are presented as mean ± standard deviation. Outliers detected and removed by Grubb’s Test (α=0.05). Comparisons between groups were assessed using two-way ANOVA with Tukey’s multiple comparison. * P<0.05, ** P<0.01, *** P<0.001, and **** P<0.0001. Figure 5. Combination PBT and α-PD-1 reduces immune suppression and elicits anti-tumor immune response in T cells. C57Bl / 6J mice were injected orthotopically into the mouse pancreas with KPC-GFP-Luc2 pancreatic tumor cells for arm 1 of the study shown in Figure 4. Two weeks later, mice were treated with PBT (0.25% DFMO in the drinking water + 4 mg / kg trimer44NMe PTI injected intraperitoneally daily), with anti-PD-1 (α-PD1) antibody (150 μg administered intraperitoneally every 2 days for 3 total doses) and / or in combination. Flow cytometry graphs depict T cell phenotypes in the spleen (Figure 5A-C) and the blood (Figure 5D-F) (n = 5 to 6 mice per group). T regs= T regulatory cells; CM=central memory; MFI=mean fluorescence intensity. Data are presented as mean ± standard deviation. Outliers detected and removed by Grubb’s Test (α=0.05). Comparisons between groups were assessed using one-way ANOVA with Tukey’s multiple comparison. * P<0.05, ** P<0.01, *** P<0.001, and **** P<0.0001. ATTORNEY DOCKET NO.10669-426PC0 Figure 6. PBT and α-PD-1 reduces myeloid-dependent immune suppression and promotes anti- tumor activity. C57Bl / 6J mice in arm 1 were injected orthotopically into the mouse pancreas with KPC- GFP-Luc2 pancreatic tumor cells. Two weeks later, mice were treated with PBT (0.25% DFMO in the drinking water + 4 mg / kg trimer44NMe PTI injected intraperitoneally daily), with anti-PD-1 (α-PD1) antibody (150 μg administered intraperitoneally every 2 days for 3 total doses) and / or in combination. Flow cytometry graphs depicting myeloid phenotypes in the spleen (Figure 6A-C) and blood (Figure 6D-F) (n = 5 to 6 mice per group). PMN-MDSCs=polymorphonuclear myeloid-derived suppressor cells; M-MDSCs=monocytic myeloid-derived suppressor cells; cDC=conventional dendritic cells. Data are presented as mean ± standard deviation. Outliers detected and removed by Grubb’s Test (α=0.05). Comparisons between groups were assessed using one-way ANOVA with Tukey’s multiple comparison. * P<0.05, ** P<0.01, *** P<0.001, and **** P<0.0001. Figure 7. Regulation of T cell activity; image from NCI website Figure 8. Structures of the native polyamines (putrescine, spermidine and spermine), polyamine- based PTIs (trimer44NMe, lasso and AMXT-1501), non-polyamine based PTIs (GW5074) and the ODC inhibitor (difluoromethylornithine, DFMO). Note: The polyamine chain is highlighted in red and provides a molecular recognition element for the polyamine transport protein ATP13A3. Figure 9. Inhibition of 4T1 tumor growth and metastasis with DFMO and Trimer PTI taken from (ref: Alexander 2020). Panel A. Mice were orthotopically injected in the mammary fat pad with 2 × 1044T1 mammary carcinoma cells. Three weeks following injection of 4T1 cells when tumors were approximately 40 mm3in size, treatment was initiated with either control vehicle or PBT (0.25% DFMO (w / v) in the drinking water plus Trimer PTI [3 mg / kg, i.p., once a day]). Graph shows 4T1 tumor growth under different treatments (mean tumor volume ± SEM). Inset shows the structures of DFMO and the Trimer PTI. Panel B. Lung metastasis were counted following perfusion of lungs with India Ink upon sacrifice (mean ± SEM). Upon sacrifice, tumors were excised from 4T1-tumor bearing mice and equal numbers of cells were analyzed by flow cytometry. Infiltrating CD45+ leukocytes analyzed for (panel C) granulocytic MDSCs (Ly6G+ / CD11b+) ± SEM, (Panel D) monocytic MDSCs (Ly6C+ / CD11b+) ± SEM and (Panel E) M2 macrophages (F4 / 80+ / CD206+) ± SEM. (Panel F) Mice were orthotopically injected in the mammary fat pad with 2 × 1044T1 mammary carcinoma cells. Two weeks later mice were injected with 100 ug of anti-Ly6G mAb or isotype control antibody (i.p., three total doses administered every other day). Three weeks following 4T1 cell injections, treatment was initiated with ATTORNEY DOCKET NO.10669-426PC0 either saline or PBT as described in earlier, and tumor growth was measured. n = 10 mice per group; * = p 0.05 and # = p 0.01 compared to vehicle treated mice. Panel G: Flow cytometry analysis of tumor- infiltrating immune cells showing decreased myeloid derived suppressor cells (MDSC), M2-like macrophages, Tregs, and increased granzyme B+IFNγ+CD8+T-cells with PBT. Figure 10. PBT alleviates doxorubicin-promoted metastasis and decreases intratumor neutrophil extracellular trap (NET) formation in 4T1 tumors. Balb / c mice were orthotopically injected in the mammary fat pad with 4T1 tumor cells and treatment initiated 2 weeks later with either saline, doxorubicin (Dox, 1.5 mg / kg, i.p., weekly), or Dox + PBT (0.5% DFMO (w / v) in the drinking water plus Trimer44NMe PTI [3 mg / kg, i.p., daily]). A) Tumor growth was measured with calipers until the mice were sacrificed 5 weeks after tumor cell injection. Mean tumor volume ± SEM. B) Number of total lung metastases as visualized after injection of India ink following euthanasia. C) Ly6G+neutrophils expressing citrullinated histone H3 (marker of NETosis) were identified by flow cytometry in the tumors (T) and spleens (S). D) Mice were orthotopically injected in the mammary fat pad with 4T1 cells. Two weeks later mice were injected with 100 ug of anti-Ly6G mAb or isotype control antibody (i.p., three total doses administered every other day). Two weeks following 4T1 cell injections, treatment was initiated with either saline or PBT, and tumor growth was measured. Figure 11. C57Bl / 6 mice with Pan02 pancreatic tumors exhibit longer median survival when treated with DFMO in combination with Trimer44NMe PTI compared to all other groups. Pan02 tumor cells (0.5 × 106) were injected into the pancreas of C57Bl / 6 mice and allowed to grow for 2 weeks. Kaplan−Meier curves for survival (n ≈ 8 for each treatment). As depicted in the chart below the graph, survival was significantly increased for mice treated with a combination of 0.25% DFMO + 1.8 mg / kg PTI compared to untreated mice (Log-rank Mantel−Cox test, p = 0.0009), but single agent treatments did not statistically increase survival compared to untreated mice (p > 0.05).21Figure 12. PBT Reduces PDAC Tumor Growth and Significantly Decreases Splenic PMN- MDSCs. KPC482GL-2 cells were orthotopically injected into the pancreas of TP53floxedmice (6-8 weeks of age). 7 days post tumor-implantation treatment, mice were treated with either vehicle, 0.25% DFMO v / w in drinking water, 4 mg / kg PTI (daily intraperitoneal injection) or a combination of the two single agents. (A) IVIS Imaging at indicated days of individual mice in each treatment group. Bioluminescence is represented as radiance and measured for total flux. (B) Quantification of IVIS imaging over the course of treatment (n = 4 mice per group). (C) Representative flow cytometry plots and graphical ATTORNEY DOCKET NO.10669-426PC0 analysis of M-MDSCs (Ly6CHighLy6G-) or PMN-MDSCs (Ly6CIntLy6G+) from CD11b+ myeloid cells isolated from spleens of treated mice (n = 3 to 4 mice per group). Data are presented as mean ± standard deviation. Comparisons between groups were assessed using one-way or two-way ANOVA with Tukey’s multiple comparison. * P<0.05, ** P<0.01, *** P<0.001, and **** P<0.0001. (D) Confirmation of KPC482GL-2 Bioluminescence Ex-Vivo. KPC482GL-2 cells were orthotopically injected into the pancreas of TP53floxed mice (6-8 weeks of age). 7 days post tumor-implantation treatment, mice were treated with either vehicle, 0.25% DFMO v / w in drinking water, 4 mg / kg PTI (daily intraperitoneal injection) or a combination of the two single agents. Mice were injected with 150 mg / kg of D-luciferin and imaged via IVIS after 10 minutes of incubation. Mice were then sacrificed and tumors and pancreas were imaged via IVIS represented as radiance and measured for total flux. Figure 13. DFMO and Trimer PTI reduced autophagy in immunosuppressive leukocytes.18Mice were orthotopically injected in the mammary fat pad with 2 × 1044T1 mammary carcinoma cells. Three weeks following injection of 4T1 cells, treatment was initiated with either control vehicle or PBT. Mice were sacrificed after three weeks of treatment, and equal numbers of cells from tumors (T), lungs (L), and spleens (S) were analyzed by flow cytometry for, A: total leukocytes (CD45+), B: autophagic activity in CD45+ leukocytes (CytoID+CD45+), C: autophagy+ MDSCs (CytoID+Gr1+CD11b+), D: autophagy+ M2 macrophages (CytoID+F480+CD206+), E; autophagy+ CD4+ T-cells (CytoID+CD4+), F: autophagy+ CD8+ T-cells (CytoID+CD8+). n = 10 per group ± SEM; * = p ≤ 0.05 and # = p ≤ 0.01 compared to vehicle treated mice. Figure 14. Inhibition of polyamine metabolism increases macrophage tumor infiltration, alters expression of FoxP3 and ATP13A3 and decreases spermine (polyamine) in the pancreatic PanO2 tumor microenvironment. Panels A-C depict images using a 40x objective (20 µm scale bar). Panel A (top) also shows images obtained with a 5x objective (500 µm scale bar). Panel A: Histology images showing increased intratumoral brown staining of F4 / 80 (a total macrophage stain) in both DFMO-treated (1% in drinking water) and DFMO+PTI trimer44NMe (1.8 mg PTI / kg) treated PanO2 tumors. Note: separate studies using mannose receptor staining suggested the relative absence of M2 macrophages in the treated pancreatic tumors or untreated tissue (data not shown). Bottom of panel A: Reduced expression of FoxP3 protein was observed in the presence of DFMO-based therapies in PanO2-challenged mice. Panel B: Relative expression of ATP13A3 protein showing increased staining in the presence of treatments consistent with an induced, increased cellular need for polyamine import. Data obtained in mice ATTORNEY DOCKET NO.10669-426PC0 challenged with PanO2 cells. Panel C: Reduced spermine (polyamine) levels in the tumor microenvironment were observed in DFMO-treated (1% DFMO in drinking water) mice challenged with PanO2 cells. Since spermine is often associated with polyribonucleic acids like DNA and RNA, respective treatments with DNAse and RNAse were used to liberate the spermine prior to antibody detection. In this manner, both the bound and unbound spermine populations can be detected. Reduced polyamine levels in DFMO-treated tissue are consistent with inhibition of polyamine biosynthesis by DFMO. It is noted that this antibody can also detect spermidine, which has also been linked to immune modulation. Figure 15. Increased T-cell infiltration in DFMO-treated pancreatic tumors. Tumor sections from PANO2-injected mice treated after one week with control, GW5074 (5 mg / kg), DFMO (0.25% w / v) and DFMO+GW5074 (PTI) for a total of four weeks. (Panel A) Representative immunohistochemistry images of CD86, CD3, CD4, CD8 and MYC-stained pancreatic tumor sections imaged at 40× magnification. Scale bars correspond to 50 µm. (Panel B) Quantification of CD86, CD3, CD4, CD8 and MYC-positive cells across treatment groups. A one-way ANOVA followed by post hoc Tukey’s test for multiple comparison was used to analyze statistical significance between treatment groups. (p < 0.05 [*], p < 0.01 [**], p < 0.001 [***], p < 0.001 [****]). This data demonstrated that specific T-cell populations are affected by DFMO and DFMO+GW5074 (the PTI parent compound). Figure 16. GW5074 versus AMXT-1501 PTI ability to reduce DFMO treated L3.6pl cell growth (72h) in high spermidine (Spd) environment in the presence of DFMO. (A) The structure of non- polyamine based PTI GW5074 and polyamine-based PTI AMXT1501. (B) GW5074 (blue bars) demonstrated consistent suppression of DFMO treated L3.6pl cell growth independent of increasing concentrations of Spd. In contrast, AMXT1501 (grey bars) demonstrated reduced ability to suppress growth of DFMO treated L3.6pl cells in the presence of increasing extracellular Spd concentration. Values represent data from experiment performed in triplicate ± S.D. **** p < 0.0001. This data suggests that non-polyamine based PTIs based on the GW5074 scaffold will likely outperform polyamine based PTIs like trimer44NMe or lasso in the presence of high polyamine concentrations in the tumor microenvironment. Figure 17. Dose dependence studies investigating3H-spermidine uptake in L3.6pl cells in the presence of each lead PTI candidate (AMXT-1501, trimer44NMe, GW5074, compounds F2, C2, and E2). ATTORNEY DOCKET NO.10669-426PC0 Figure 18. Left panel: Dose Dependence Experiments with F2 demonstrating inhibition of14C- spermine (14C-Spm) entry into L3.6pl cells over 15 min. These responses were used to estimate the14C- Spm IC50 values. Right panel: Dose Dependence Experiments with F2 showing a lack of inhibition of3H-putrescine (3H-Put) entry into L3.6pl cells over 15 min incubation period. This demonstrated that compound F2 was a specific inhibitor of spermidine and spermine uptake and not putrescine at the concentrations tested. Figure 19. C57Bl / 6 mice (n = 5-7 mice per treatment group) were orthotopically injected with 1 x 105EO771.LMB tumor cells in the mammary fat pad and then administered ICB (100 µg anti-PD-1 mAb or 100 µg control isotype mAb) on days 10, 13, and 17. On day 12, daily treatment was initiated with either vehicle control or PBT (0.5% DFMO in the drinking water + 3 mg / kg trimer44NMe PTI, i.p., daily). Tumor growth was measured with calipers until mice with tumors were sacrificed 5 weeks after tumor cell injection. Treatment with PBT was stopped for 3 additional mice treated with PBT + PD-1 mAb whose tumors completely regressed. When no tumor growth was observed over the next 3 weeks, these mice were rechallenged with an orthotopic injection of 1 x 105EO771.LMB tumor cells in a mammary fat pad on the opposite side. The mice were observed for secondary tumor growth with no further treatment for 2 months. Figure 20. Shown is a graph that was produced after running a linear mixed effects model with an interaction between time and the treatment groups (time*groups). The predicted log tumor volumes were then graphed at each time point. The p < .0001 indicates that the interaction is significant, meaning that there is evidence of a synergistic effect. Figure 21. Bliss Combination Index (CI) shows how the combination of PD-1 + PBT compares to what one would expect if the drugs simply added up (no interaction effect). The dotted line at 1.0 is the cutoff between synergy and antagonistic or additive effects. The Bliss Synergy Score (i.e. Bliss independence) shows how much better the combination therapy works than one would expect if both drugs were working independently. A score greater than zero shows synergy. At each time point, the combination therapy shows a synergistic effect for both CI and Synergy Score. Figure 22. HSA Combination Index (CI) shows how the combination of PD-1 + PBT compares to the most effective single treatment (either PD-1 or PBT). The dotted line at 1.0 represents an additive ATTORNEY DOCKET NO.10669-426PC0 effect — meaning the combination is no better than the better monotherapy. Values below 1.0 indicate synergy, where the combination outperforms the best single agent. Figure 23. Doxorubicin enrichment of slow-cycling ATP13A3+tumorspheres (A) Number of viable PKH26-labeled SUM159 tumorsphere cells following 3 days culture ± 25 nM DOX ± 1 uM Trimer PTI or AP, another PTI. (B) SUM159 tumorspheres treated 5 days ± 25 nM DOX, then non- permeabilized cells analyzed by flow cytometry for % of viable cells with surface expression of PKH26, ATP13A3, CD304, CD243, SETDB1, and for autophagic flux following staining with CytoID. Figure 24. EO771ATP13A3-FDtumorspheres are more sensitive to cytotoxic doxorubicin and to PBT than parental EO771 cells. Equal numbers of parental EO771.lmb TNBC cells (blue bars) and EO771.lmb cells with a functionally dead ATP13A3 (red bars) were cultured in ultra-low adherence plates in serum-free medium to form tumorspheres and treated for 3 days ± 1 nM or 6 nM doxorubicin (Dox) ± PBT ( 0.6 mM DFMO + 1 μM Trimer PTI; striped bars). Tumorspheres were analyzed for viable cells by flow cytometry. Figure 25. Equal numbers of L3.6pl-ATP13A3WTcells (solid bars) and L3.6pl-ATP13A3FDcells (striped) were cultured in ultra-low adherence plates in serum-free medium to form tumorspheres and treated for 3 days ± 1 μM gemcitabine (Gem, in red) ± 5 μM Trimer PTI ± 50 μM spermidine. Tumorspheres were analyzed for viability by flow cytometry. Figure 26. Boyden chamber inserts with 8 µm porous filters were coated with growth factor- reduced Matrigel and then seeded with 1x105parental EO771.lmb tumor cells (blue bar) and EO771.lmb cells with a functionally dead ATP13A3 (red bar) in serum-free media in the well of each insert. A gradient was then initiated with the addition of serum-free media supplemented with NIH3T3 conditioned medium in the lower compartment of the invasion chamber. After a 24-hour incubation, cells that had invaded to the underside of the filter were fixed, stained, and counted using a microscope. Data expressed as mean number of cells ± SD. Figure 27. Parental EO771 tumor cells and mutant EO771ATP13A3-FDtumor cells (1 x 105) were orthotopically injected into the mammary fat pad of C57Bl / 6 mice. Mice were treated 1% DFMO in drinking water or water alone starting when tumors were 30-50 mm3in size (n = 4-5 mice / treatment group). ATTORNEY DOCKET NO.10669-426PC0 Figure 28. Mutant EO771ATP13A3-FDtumor cells (1 x 105) were orthotopically injected into the mammary fat pad of C57Bl / 6 mice. Mice were treated 3 times with anti-PD-1 mAb (ICB) or isotype control mAb followed by treatment with 0.5% DFMO in their drinking water or water alone starting when tumors were 30-50 mm3in size (n = 6-7 mice / treatment group). Treatment was stopped for mice whose tumors completely regressed by day 22 of treatment, and 3 weeks later mice with completely regressed tumors were challenged with an orthotopic injection of parental EO771 tumor cells (1 x 105) into the mammary fat pad on the opposite side. * No secondary tumor growth was observed in any re- challenged mouse over the next 2 months with no further treatment. Figure 29. Neutrophils from healthy volunteers were stimulated for 1 hr with 100 nM PAF to undergo NETosis. NETs were visualized following staining with PICO green or immunofluorescent staining with an antibody against citrullinated histone 3. Figure 30. Neutrophils from healthy volunteers were stimulated to undergo NETosis for 2 hr with 150 nM TPA ± 1 μM Trimer polyamine transport inhibitor. NETs were visualized following staining with PICO green. Figure 31. Pico Green fluorescent staining of bone marrow-derived neutrophils (more than 95% Ly6GHighby flow cytometry and having the typical segmented nuclei of mature neutrophils by microscopy) stimulated for 3 hr with A) 100 nM PMA; and B) 100 nM PMA + 1 uM Trimer PTI. C) Neutrophils were stimulated for 30 min with control vehicle or 100 nM PMA. Nonpermeabilized neutrophils were then analyzed by flow cytometry for surface expression of ATP13A3 and citrullinated histone H3 (NETosis marker). No NETs detected at this early time point. Figure 32. Survival Curve for WT C57Bl / 6 & PAD4 KO Mice Treated with DOX ± PBT. Nontumor-bearing wildtype (WT) C57Bl / 6 mice and PAD4 KO mice were treated twice a week with 4 mg / kg Doxorubicin (DOX) for 3 weeks. Half the mice were also treated with Polyamine Blocking Therapy (PBT, 0.5% DFMO in the drinking water and 3 mg / kg Trimer PTI, ip daily) during the 3 weeks of DOX treatment. DESCRIPTION The embodiments described herein are based on the use of the combination therapy involving an inhibitor of polyamine biosynthesis (like difluoromethylornithine, DFMO) along with a polyamine ATTORNEY DOCKET NO.10669-426PC0 transport inhibitor (PTI) to starve cancer cells of the polyamine resources they need to grow (Figure 1). This approach of DFMO+PTI is known as polyamine blocking therapy (PBT) and can be used to curb the growth of rapidly-proliferating cell types such as cancers and certain infectious diseases. As disclosed herein, PBT has a profound effect on the immune system and potentiates immune checkpoint inhibitors such as programmed cell death protein-1 (PD-1) inhibitors. Current immune checkpoint inhibitors and the companies that sell them include but are not limited to: Keytruda (Pembrolizumab, anti-PD-1, Merck), Tecentriq (Atezolizumab; anti-PD-L1, Roche and Chugai Pharmaceuticals), Libtayo (Cemiplimab, anti-PD-1, Regeneron) and Durvalumab (Imfinzi). Immune Checkpoint Inhibitors in the Clinic 1. Anti-PD-1 Pembrolizumab (Keytruda, Merck) Nivolumab (Opdivo, Bristol-Myers Squibb and ONO Pharmaceuticals) Cemiplimab (Libtayo, Regeneron-Sanofi) 2. Anti-CTLA-4 Ipilimumab (Yervoy, Bristol-Myers Squibb) 3. Anti-PD-L1 Atezolizumab (Tecentriq, Roche and Chugai Pharmaceuticals) Avelumab (Bavencio, Merck KGaA and Pfizer) (for metastatic Merkel-cell carcinoma) Durvalumab (Imfinzi, AstraZeneca) The foregoing immunotherapies would all benefit from a combination therapy that potentiates compounds for treatment in cancers which are recalcitrant to immune checkpoint inhibitors like triple negative breast cancers and pancreatic cancers. In some aspects, PBT, DFMO and / or PTI are co- administered with an immune checkpoint inhibitor that inhibits an immune checkpoint protein or ligand thereof selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or adenosine A2a receptor (A2aR). In certain aspects, the immune checkpoint inhibitor is a PD-1 inhibitor. In some aspects, the PD-1 inhibitor is nivolumab, pembrolizumab, CT-011, BMS 936559, MPDL328OA or AMP-224. In certain aspects, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In particular aspects, the CTLA-4 inhibitor is ipilimumab or tremelimumab. In ATTORNEY DOCKET NO.10669-426PC0 certain embodiments, as will be described herein, involve the administration of PBT in conjunction with administration of one or more immune checkpoint inhibitors. These and many other embodiments are further described herein. Definitions The terms “administering” or “administer” or “administration” as used herein with respect to an agent means providing the agent to a subject using any of the various methods or delivery systems for administering agents or pharmaceutical compositions known to those skilled in the art. Modes of administering include, but are not limited to oral administration, parenteral administration such as intravenous, subcutaneous, intramuscular or intraperitoneal injections, rectal administration by way of suppositories, transdermal administration, intraocular administration or administration by any route or method that delivers a therapeutically effective amount of the drug or composition to the cells or tissue to which it is targeted. Alternatively, routine experimentation will determine other acceptable routes of administration. As used herein, an “adjunct cancer therapeutic agent” pertains to an agent, other than polyamine transport inhibitors, polyamine synthesis inhibitors, or immune checkpoint inhibitors described herein, that possesses selectively cytotoxic or cytostatic effects to cancer cells over normal cells. As used herein, the term "adjunct cancer therapy protocol" refers to a therapy, such as surgery, chemotherapy (e.g. administering adjunct cancer therapeutic agent), radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect when administered in conjunction with administration of a polyamine transport inhibitor, polyamine synthesis inhibitor, and / oror immune checkpoint inhibitor. Such beneficial effects include reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer. Cytostatic and cytotoxic agents that target the cancer cells are specifically contemplated for combination therapy. Likewise, agents that target angiogenesis or lymphangiogenesis are specifically contemplated for combination therapy. As used herein, the terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to, lung cancers, including small cell ATTORNEY DOCKET NO.10669-426PC0 lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung; bladder cancers (e.g., urinary bladder cancer (UBC), muscle invasive bladder cancer (MIBC), and BCG- refractory non-muscle invasive bladder cancer (NMIBC)); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); cancer of the urinary tract; breast cancer (e.g., HER2+ breast cancer and triple- negative breast cancer (TNBC), which are estrogen receptors (ER−), progesterone receptors (PR−), and HER2 (HER2−) negative); prostate cancer, such as castration-resistant prostate cancer (CRPC); cancer of the peritoneum; hepatocellular cancer; gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; hepatoma; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine carcinoma; salivary gland carcinoma; prostate cancer; vulval cancer; thyroid cancer; hepatic carcinoma; anal carcinoma; penile carcinoma; melanoma, including superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanomas, and nodular melanomas; multiple myeloma and B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myelogenous leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal vascular proliferation associated with phacomatoses, edema (such as that associated with brain tumors), Meigs' syndrome, brain cancer, head and neck cancer, and associated metastases. The term “co-administer” or other grammatical forms thereof, as used herein refers to the administration of an active agent before, concurrently, or after the administration of another active agent such that the biological effects of either agents overlap. It is noted that terminology used herein of administering an agent “in combination with” another agent is to be understood as co-administration. As used herein, the term “immune checkpoint inhibitor” refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins. As used herein, the term “immune checkpoint protein” has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are ATTORNEY DOCKET NO.10669-426PC0 recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. 2011. Nature 480:480-489). Examples of stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, OX40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor- specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T-Lymphocyte- Associated protein 4 and also called CD152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme. A related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. KIR, Killer-cell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells. LAG3, Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. PD-1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. TIM- 3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 acts as a negative regulator of Th1 / Tc1 function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection ATTORNEY DOCKET NO.10669-426PC0 by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti-tumor T-cell response. In some embodiments, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands. In a particular embodiment, the immune checkpoint inhibitor is an antibody. Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA. In a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, WO2006121168, WO2015035606, WO2004056875, WO2010036959, WO2009114335, WO2010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK). In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody such as described in WO2013079174, WO2010077634, WO2004004771, WO2014195852, WO2010036959, WO2011066389, WO2007005874, WO2015048520, U.S. Pat. No. 8,617,546 and WO2014055897. Examples of anti-PD-L1 antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech / Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS). In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in U.S. Pat. Nos. 7,709,214, 7,432,059 and 8,552,154. In the context of the invention, the immune checkpoint inhibitor inhibits Tim-3 or its ligand. In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and WO2013006490. In some embodiments, the immune checkpoint inhibitor is a small organic molecule. ATTORNEY DOCKET NO.10669-426PC0 The term “small organic molecule” as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA. In a particular embodiment, small organic molecules interfere with transduction pathway of PD- 1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway. In a particular embodiment, the small organic molecules interfere with Indoleamine-pyrrole 2,3- dioxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), β-(3-benzofuranyl)-alanine, β-(3- benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6-fluoro-tryptophan, 4-methyl-tryptophan, 5-methyl tryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5-hydroxy-tryptophan, indole 3-carbinol, 3,3′- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3-Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a β-carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1- methyl-tryptophan, β-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-Amino-naphtoic acid and β-[3- benzo(b)thienyl]-alanine or a derivative or prodrug thereof. In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to —N-(3-bromo-4-fluorophényl)-N′-hydroxy-4- {[2-(sulfamoylamino)-éthyl]amino}-1,2,5-oxadiazole-3 carboximidamide: In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in WO2009054864, refers to 1H-1,2,4-Triazole-3,5-diamine, 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2- c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(1-pyrrolidinyl)-5H-benzocyclohepten-2-yl]- and has the following formula in the art: ATTORNEY DOCKET NO.10669-426PC0 In a particular embodiment, the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA-170 are presented by Curis Collaborator and Aurigene on November at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics. In some embodiments, the immune checkpoint inhibitor is an aptamer. Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA. In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti- PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015. The term polyamine transport inhibitor or “PTI” as used herein refers to a compound that inhibits or reduces the uptake of polyamines into cells. Polyamines are essential for cell growth and division, and cancer cells often rely on increased polyamine levels. The terms “synergy”, “synergistically” or “synergistic effect” is intended to have its ordinary meaning to one of skill in the art. A synergistic effect produced by a combination of compounds can include an effect which is observed to be greater than the effect produced by each compound individually. For example, in the case of PBT and immune checkpoint inhibitors synergistically decrease cell viability of cancer cells when used in combination to treat a cancer, the Polyamine transport inhibitor, polyamine synthesis inhibitor and an immune checkpoint inhibitor have a mutual enhancing of the effect of each individual compound by the combination of compounds that produces more than additive effect. In certain instances, two or more compounds having a synergistic effect allows for lower doses of the individual compounds when co-administered to produce the same or greater therapeutic effect or outcome compared to higher doses of the compound administered singly. ATTORNEY DOCKET NO.10669-426PC0 The terms “subject,” “individual,” “host,” and “patient,” are used interchangeably herein to refer to an animal being treated with one or more enumerated compounds as taught herein, including, but not limited to, simians, humans, avians, felines, canines, equines, rodents, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets. A suitable subject for the invention can be any animal, preferably a human, that is suspected of having, has been diagnosed as having, or is at risk of developing a disease that can be ameliorated, treated or prevented by administration of one or more enumerated compounds. The term “treating” or “treatment of” as used herein refers to providing any type of medical management to a subject. Treating includes, but is not limited to, administering a composition comprising one or more active agents to a subject using any known method for purposes such as curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition or one or more symptoms or manifestations of a disease, disorder or condition. A “therapeutically effective amount” refers to an amount which, when administered in a proper dosing regimen, is sufficient to reduce or ameliorate the severity, duration, or progression of the disorder being treated (e.g., cancer), prevent the advancement of the disorder being treated (e.g., cancer), cause the regression of the disorder being treated (e.g., cancer), or enhance or improve the prophylactic or therapeutic effects(s) of another therapy. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations per day for successive days. Overview The data in the present disclosure demonstrates the improved outcomes when polyamine blocking therapy, or “PBT” (DFMO+PTI) is combined with an immune checkpoint inhibitor (e.g., PD-1 inhibitor, CTLA-4, PD-L1, and PD-L2). Figure 1 provides a general diagram of the cellular mechanisms involved in the this combined therapy (for example: DFMO+PTI+PD-1 inhibitor). The data in Figure 2 shows the combined therapy results in increased anti-tumor and anti-metastatic activity and significantly increased survival in mouse models of both triple negative breast cancer (4T1 and EO771.lmb tumor cells) and melanoma (B16F10 tumor cells) that are resistant to the PD-1 inhibitor alone (Figure 2A-C). ATTORNEY DOCKET NO.10669-426PC0 PBT sensitizes resistant tumors to PD-1 blockade and re-programs T-cells into an anticancer phenotype resulting in smaller tumors and increased survival. (Figure 2A). In addition, the data herein demonstrates a number of cures that have been observed in mouse models of triple negative breast cancer (TNBC) using EO771.lmb tumor cells. Indeed, Figure 2D demonstrates a durable cure in a mouse model of resistant TNBC following treatment with PBT + PD-1 blockade (3 / 5 cured,) and in pancreatic cancer models using KPC pancreatic tumor cells (3 / 4 cured, Figure 3). Importantly, this approach imparts immune cell memory so that when a mouse with a completely regressed tumor is rechallenged with these same tumor cells three weeks later, there is no tumor growth indicating a durable anti-tumor immune memory (Figure 2D). The therapeutic approaches disclosed herein sensitizes recalcitrant cancer cell lines to immune checkpoint inhibitors like PD1 inhibitors. Further, the administration of certain compounds (e.g. DFMO and / or PTI) described herein can be expanded to include other immune modulating agents and approaches (e.g., CTLA-4, PD-L1, and PD-L2) with PD-1 inhibition being preferred. The idea is that the polyamine targeting therapies (DFMO and PTI) reprogram the immune cells to be more anti-tumor in phenotype (where both M2 macrophages and myeloid derived suppressor cells are reduced in number). These events coupled with the more aggressive T cells (due to PD-1 inhibition) and improved immune memory work together to defeat and clear the tumor cells. As disclosed herein, the orthotopic KPC pancreatic ductal adenocarcinoma (PDAC) mouse model was refined that in one embodiment involved specifically resuspending tumor cells in BME (a Matrigel-like basal membrane extract) prior to injection into the pancreas. In brief, C57Bl / 6J mice (~6 weeks of age) were orthotopically injected with 0.5 × 10⁶ KPC-GFP-Luc2 pancreatic tumor cells (Figure 4A). Mice with confirmed tumor engraftment were randomized into treatment groups consisting of PBS (control), PBT, anti-PD-1, or combination PBT + anti-PD-1 therapy (n = 5–6 mice per group), and a duplicate arm was performed to show reproducibility. Mice underwent an expanded 28-day treatment period. Overall tumor progression was followed using bioluminescent imaging via IVIS in vivo imaging system (Figure 4B), and it demonstrated significant tumor repression over the entire experiment when mice were treated with combination PBT + anti-PD-1. ATTORNEY DOCKET NO.10669-426PC0 The flow cytometry analysis of mice was expanded in arm 1 of the study from Figure 4 to more comprehensively evaluate the immune modulatory effects of combination therapy, specifically on T cell populations (Figure 5). Analysis of the T cell compartment revealed a reduction in the prognostic CD4 / CD8 ratio in both blood (Figure 5A) and spleen (Figure 5D), suggesting systemic mitigation and potential shifting of T cell subsets and / or immunosuppression with the combination treatment of anti- PD-1 and PBT compared to the control group. Also, the comparison between these treatments showed trending reduction in naïve CD4⁺ T cells, a population associated with poor PDAC outcomes in spleen (Figure 5B) and especially in blood (Figure 5E). Evaluation of the blood also showed a similar trend for reduction of CD4+ T regulatory cells (CD25+FOXP3+) in the combination treatment compared to the control group (Figure 5E). An interesting finding was that certain CD8⁺ T cell (CD3⁺CD45⁺CD8⁺) populations showed promising shifts, with a significant increase in naïve (CD44⁻CD62L⁺) and central memory (CD44⁺CD62L⁺) CD8⁺ T cells in the spleen (Figure 5C). These findings support the emergence of a functional and persistent anti-tumor T cell repertoire. CD25 expression, a marker associated with T cell memory potential, was also found to be significantly increased on CD8⁺ T cells in the spleen, further supporting a shift toward durable anti-tumor immunity (Figure 5C). Similar trends were observed for comparisons between the combination treatment compared to the control group in the blood (Figure 5F). Changes in the myeloid cell populations were also observed via flow cytometry analysis of the spleens and peripheral blood from arm 1 of the 28-day study in Figure 4. PMN-MDSCs (CD45⁺CD11b⁺Ly6GhighLy6Cint) were significantly decreased in both spleen (Figure 6A) and blood (Figure 6D) for mice treated with the anti-PD-1 + PBT combination compared to PBT and / or control group. Additionally, M2 macrophages were reduced in the spleens of anti-PD-1 and / or combination- treated animals compared to the control group (Figure 6B). This was not as apparent in the blood since macrophages are more abundantly found in the spleen (Figure 6E). The analysis was further expanded to assess dendritic cell populations. Although the total number of dendritic cells (CD45+F4 / 80-CD11c+) in blood and spleen showed a trend for reduced cells with the anti-PD-1 and combination with PBT compared to the control group, the results showed that certain dendritic subtypes exhibited significant shifts. A marked decrease in conventional dendritic cell type 2 (cDC2) (CD11b+), an immunosuppressive population, and a corresponding increase in cDC1 (CD11b-), which is associated with effective antigen presentation and anti-tumor activity, was observed in spleens and blood from combination treated compared to PBT and / or control group mice (Figures 6C, 6F). These findings ATTORNEY DOCKET NO.10669-426PC0 collectively support the conclusion that combination therapy remodels the immune environment toward a more effective anti-tumor phenotype. As shown in Figure 7, T cell response can be modulated by targeting T cells and tumor cells via anti-PD-1 or anti-PD-L1 for example. Even though polyamines are an established anti-proliferative target, strategies to inhibit polyamine pools often fail. For example, efforts to inhibit the polyamine biosynthetic enzymes (ornithine decarboxylase (ODC), spermidine synthase (SRM) and spermine synthase (SMS) in human cancers often induce intracellular polyamine depletion but have limited effect on growth unless the total polyamine pools are decreased dramatically.1For example, in pancreatic ductal adenocarcinoma (PDAC) cells (L3.6pl cells) one needed to reduce the intracellular polyamine pools below 80% of the untreated control to affect growth in cell culture.2In this regard, cancers have excess pools of polyamines to accommodate / survive any temporary reduction of polyamine pools. Indeed, treatment of L3.6pl human pancreatic cancer cells with polyamine biosynthesis inhibitors (e.g., alpha- difluoromethylornithine (DFMO, an ODC inhibitor), trans-4-methylcyclohexylamine (MCHA, a spermidine synthase inhibitor) or N-cyclohexyl-1,3-diaminopropane (CDAP, a spermine synthase inhibitor) caused a reshuffling of intracellular polyamine pools and little to no effect on cell growth, except for the DFMO-treated entries.1In this regard, ODC is a preferred target as its inhibition affects cell growth. The problem with targeting ODC is that ODC protein has a short half-life in humans (~30 min) and therefore requires high doses of the inhibitor (DFMO) in order to be effective.3The good news here is that DFMO (Figure 1) has a 40 year history in the clinic, is well tolerated by patients and is non-toxic even at high doses (500-1500 mg / m2 / day).3-5DFMO is already approved for the treatment of African sleeping sickness (i.e., African trypanosomiasis). The bad news is that DFMO-treated tumors often circumvent the block of polyamine biosynthesis (i.e. the inhibition of ODC) by importing extracellular polyamines to make up the polyamine pool deficiency.2For this reason, disclosed herein is the development of novel polyamine transport inhibitors (PTIs) to address this tumor escape pathway.6-13Together the combination of DFMO and the PTI has been called polyamine blocking therapy (PBT) because it provides a ‘full-court press’ on polyamine metabolism by inhibiting both polyamine biosynthesis and polyamine import. Polyamine-based2and non-polyamine-based polyamine transport ATTORNEY DOCKET NO.10669-426PC0 inhibitors11,14, 15have been developed (Figure 8) to be used in combination with DFMO. Indeed, PBT has given rise to improved outcomes in several cancer model systems including melanoma, breast cancer, neuroblastoma,11, 16colorectal, and pancreatic cancers.6, 9, 10, 13-15, 17-23In other embodiments, polyamine transport inhibitor(s) is / are co-administered, without the need for DFMO, in combination with an immune checkpoint inhibitor. In a specific embodiment, a PTI is co- administered with a PD1 inhibitor. In a specific embodiment, the PTI(s) in combination with a PD1 inhibitor is directed to the following PTIs: As shown in Figure 9A, PBT has efficacy against the 4T1 model of metastatic triple negative breast cancer.18Smaller tumors were associated with changes in immune cell populations indicating that PBT was having an effect on the tumor microenvironment and immune response. In recent unpublished work, it is demonstrated that PBT alleviates doxorubicin-promoted metastasis and decreases intratumor neutrophil extracellular trap (NET) formation in 4T1 tumors (Figure 10). This finding suggests that PBT can be used with existing chemotherapeutics like doxorubicin to improve outcomes (reduce lung metastases and reduce relative tumor size). As disclosed herein, the efficacy of PBT (combination of trimer44NMe PTI + DFMO) was demonstrated in pancreatic cancers using PanO2 murine pancreatic cancer cells in an orthotopic model ATTORNEY DOCKET NO.10669-426PC0 (Figure 11)21and recently showed a similar result using isolated KPC cells (KPC482GL-2) with the combination of trimer44NMe PTI + DFMO (Figure 12). As shown in Figure 12, smaller tumors were observed in the PBT treated arm and a significant reduction was observed in polymorphonuclear- myeloid derived suppressor cells (PMN-MDSC) cells in the presence of PBT. As such, changes in this unique cell population could provide a marker of PBT efficacy in peripheral blood. Note: the changes in M-MDSCs was less significant. In summary, PBT demonstrates efficacy against several cancer types: melanoma, breast cancer, neuroblastoma, colorectal, and pancreatic cancers.6, 9, 10, 13-15, 17-23Immune modulation. PBT has a profound effect on the immune cell population18as illustrated in Figure 13. PBT changes macrophage populations in vivo as well as expression of the polyamine transport protein ATP13A324, 25and the levels of the polyamine spermine in the tumor microenvironment (Figure 14). As shown in Figure 15, changes in other immune cell types in a pancreatic cancer model are demonstrated including T cells in the presence of PBT. Collectively, the present disclosure provides compelling evidence that polyamine blocking therapy modulates the immune response (Figures 9-15) and that combining this therapy with other immunotherapies has surprisingly effective outcomes (Figures 2 and 3). In particular, the present disclosure shows PBT in combination with other immunotherapies results in cures in both triple negative breast cancers and pancreatic cancer murine models. Description of Exemplary Embodiments In certain embodiments, provided are methods for the manufacture of compound F2, C2, and E2. In other embodiments, disclosed are the use of F2, C2, and / or E2 as a PTI in the treatment of proliferative diseases with cancers being preferred. F2, C2, and / or E2 may be used in combination with a polyamine biosynthesis inhibitor (difluoromethylornithine, DFMO) for the treatment of proliferative diseases with cancers being preferred. In a specific embodiment, provided is the use of F2 , C2, and / or E2 in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of cancer where F2, C2, and / or E2 + DFMO provide an immunotherapy adjuvant which potentiates another immunotherapeutic agent such as a PD1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor or PD2 inhibitor ATTORNEY DOCKET NO.10669-426PC0 with a PD1 inhibitor being preferred. In specific embodiments, the cancer is breast cancer (e.g. triple negative breast cancer), colorectal, pancreatic cancer, and / or melanoma. According to other embodiments, disclosed is the use of trimer44NMe or its pharmacologically acceptable salt as a PTI in the treatment of proliferative diseases such as cancer. In a more specific embodiment, provided is use of trimer44NMe or its pharmacologically acceptable salt in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of cancer where trimer44NMe + DFMO provide an immunotherapy adjuvant which potentiates another immunotherapeutic agent such as a PD1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor or PD2 inhibitor with a PD1 inhibitor being preferred. In specific embodiments, the cancer is breast cancer (e.g. triple negative breast cancer), colorectal, pancreatic cancer, and / or melanoma. In other related embodiments, disclosed is a combination therapy including a polyamine biosynthesis inhibitor and a polyamine transport inhibitor as an immune modulating therapeutic in the treatment of cancers with breast, colorectal, melanoma and pancreatic cancers. Combination therapy refers to the co-administration of the named compounds. In a specific embodiment, combination therapy includes co-administering difluoromethylornithine (DFMO) and a polyamine transport inhibitor and a PD-1 inhibitor for use in the treatment of cancers with breast, colorectal, melanoma and pancreatic cancers being preferred. In further embodiments, provided is the use of difluoromethylornithine and a polyamine transport inhibitor in combination with existing chemotherapeutic agents such as doxorubicin in the treatment of cancers. Moreover, other embodiments pertain to the use of a PBT (polyamine transport inhibitor (e.g. trimer44NMe PTI) and a polyamine biosynthesis inhibitor (e.g.DFMO)) as an immune adjuvant agent to boost the efficacy of an immunotherapeutic agent which has shown insufficient of cancer when administered alone. In further embodiments, disclosed is the use of PBT to metabolically reprogram immune cells to increase tumor sensitivity to an immunotherapeutic agent, wherein, optionally, the immunotherapeutic agent comprises at least one immune checkpoint inhibitor. ATTORNEY DOCKET NO.10669-426PC0 Further embodiments pertain to a method of treating cancer comprising co-administering PD-1 blockade and polyamine blocking therapy (PBT) in a synergistic amount, wherein the synergistic amount achieves a cancer cell killing effect at lower doses than what is achievable by administering PD- 1 blockade or PBT alone. In another embodiment, disclosed is a method for treating chemoresistant cancer exhibiting increased ectopic expression of ATP13A3, the method comprising administering a therapeutically effective amount of PBT, wherein the therapeutically effective amount inhibits ATP13A3 expression or activity. In yet another embodiment, provided is a method comprising co-administering an adjunct cancer therapy protocol with PBT or PTI without DFMO at least one immune checkpoint inhibitor to achieve a durable anti-tumor immune response and immune memory in tumor that is being concurrently treated therewith but is resistant to immune checkpoint blockade. Further still, provided is a method for inhibiting NETosis and NET-stimulated tumor progression and metastasis, wherein the method comprises administering a therapeutically effective amount of a trimer PTI, either alone or in combination with a chemotherapeutic agent like doxorubicin. Another embodiment pertains to a method comprising administering PBT to a subject in need, wherein the subject in need exhibits Doxorubicin-induced cardiotoxicity. Reference to a use of a compound or combination therapy comprises administering a therapeutically effective amount of the named compound or compounds. Combination therapy may comprise co-administering a therapeutically effective amount of each of the named compound or compounds to treat cancer. In another embodiment, disclosed is a method of treating cancer comprising co-administering an immune checkpoint inhibitor and polyamine blocking therapy (PBT) in a synergistic amount, wherein the synergistic amount achieves a cancer cell killing effect at lower doses than what is achievable by administering an immune checkpoint inhibitor or PBT alone. Another embodiment pertains to a method of treating cancer in a subject comprising co- administering a therapeutically effective amount of an immune checkpoint inhibitor and a therapeutically effective amount of a polyamine blocking therapy (PBT) to the subject. ATTORNEY DOCKET NO.10669-426PC0 Compositions, Formulations and Modes of Delivery In certain embodiments, DFMO, PTIs or immune checkpoint inhibitors are formulated in singular compositions or in compositions containing a combination of such compounds. In certain embodiments the compounds described herein are provided as pharmaceutically acceptable salts. For instance, pharmaceutically acceptable salts of the compounds provided herein are synthesized from the parent compounds, which contain a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. Generally, nonaqueous media like ether, ethyl acetate, ethanol, 2-propanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N- dialkylenethanolamine, triethanolamine and basic amino acids salts. In addition, any compound referred to herein may be in crystalline or amorphous form either as free compounds or as solvates (e.g. hydrates) and it is intended that all forms are within the scope of the present invention. Methods of solvation are generally known within the art. Pharmaceutical compositions comprising DMFO, and / or PTI, and / or an immune checkpoint inhibitor or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier may be formulated according to the chosen route of administration. Examples of the administration form include without limitation oral, topical, parenteral, sublingual, rectal, vaginal, ocular and intranasal. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Preferably the compositions are administered parenterally. Pharmaceutical compositions can be formulated so as to allow a compound to be bioavailable upon administration of the composition to an animal, preferably human. Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of a compound may contain the compound in liquid or in aerosol form and may hold a single or a plurality of dosage units. ATTORNEY DOCKET NO.10669-426PC0 The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) can be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) can be gaseous, or liquid so as to provide an aerosol composition useful in, for example inhalatory administration. Powders may also be used for inhalation dosage forms. The term “carrier” refers to a diluent, adjuvant or excipient, with which the compound according to the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, disaccharides, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the compounds and compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the compounds are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid. As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more for the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agent such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. ATTORNEY DOCKET NO.10669-426PC0 When the composition is in the form of a capsule (e.g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrins or a fatty oil. The composition can be in the form of a liquid, e.g. an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye / colorant and flavour enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included. The preferred route of administration is parenteral administration including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intracerebral, intraventricular, intrathecal, intravaginal or transdermal. The preferred mode of administration is left to the discretion of the practitioner, and will depend in part upon the site of the medical condition. In a more preferred embodiment, the compounds according to the present invention are administered intravenously. Infusion times of up to 24 hours are preferred to be used, more preferably 1 to 12 hours, with 1 to 6 hours being most preferred. Short infusion times which allow treatment to be carried out without an overnight stay in a hospital are especially desirable. However, infusion may be 12 to 24 hours or even longer if required. Infusion may be carried out at suitable intervals of, for example, 1 to 4 weeks, preferably once every three weeks. Liquid compositions, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides, polyethylene glycols, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material. Physiological saline is a preferred adjuvant. The compositions comprise an effective amount of DMFO, and / or PTI and / or an immune checkpoint inhibitor such that a suitable dosage will be obtained. The correct dosage will vary according ATTORNEY DOCKET NO.10669-426PC0 to the particular formulation, the mode of application, and its particular site and host. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease should be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose. The dose will be selected according to the dosing schedule, having regard to the existing data on preferred administration routes and dosages for each compound. In specific embodiments, it can be desirable to administer DMFO and / or PTI and / or an immune checkpoint inhibitor locally to the area in need of treatment. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumour or neoplastic or pre-neoplastic tissue. Pulmonary administration can also be employed, e.g. by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, DMFO and / or PTI and / or immune checkpoint inhibitor can be formulated as a suppository, with traditional binders and carriers such as triglycerides. The present compositions can take the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. The pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining DMFO and / or PTI and / or immunecheckpoint inhibitor with water, or other physiologically suitable diluent, such as phosphate buffered saline, so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension. Example 1: Testing of Non-Polyamine based PTIs As shown in Figure 16, the use of non-polyamine based PTIs may be especially effective as they are insensitive to exogenous polyamine concentrations which are likely high in the tumor microenvironment (Figure 14, panel C). Figure 16 shows that the non-polyamine based PTI GW507415is insensitive to added spermidine (a polyamine), whereas the AMXT-1501 PTI (Aminex Therapeutics) ATTORNEY DOCKET NO.10669-426PC0 loses its potency in the presence of high spermidine concentrations. Table 1 provides data indicating the IC50 values of different tested PTIs. Table 1. IC50values of top performing PTIs in the3H-spermidine (3H-Spd) uptake assay in L3.6pl cellsaPTI Name PTI structure3H-Spd ATTORNEY DOCKET NO.10669-426PC0 Since the indolone compound F2 (CAS 220904-61-0) outperformed the known parent GW5074 PTI (Tables 1 and Figure 17) it represents a novel non-polyamine based PTI as well as compounds C2 (CAS 1608092-89-2) and E2 (Z isomer: CAS 220904-99-4 and E isomer: CAS 1191386-68-1). A dose dependent study of these PTIs agents was performed to derive the IC50values in Table 1. Figure 17 provides data of dose dependence studies investigating3H-spermidine uptake in L3.6pl cells in the presence of each lead PTI candidate (AMXT-1501, trimer44NMe, GW5074, compounds F2, C2, and E2). The F2 compound also targets spermine uptake, but not putrescine uptake in L3.6pl pancreatic cancer cells (Figure 18). Example 2: General Synthetic Procedure for indolone compounds C2, E2 and F2 Piperidine (0.2 eq) and substituted benzaldehyde (1.0 eq) was added to a solution of substituted indolone (1.0 eq) in ethanol (0.25M). The reaction refluxed overnight at 95oC. After cooling to room temperature, the reaction mixture was filtered and washed with ethanol. Recrystallization of the precipitate in methanol resulted in a bright yellow solid product. C2:1H NMR (DMSO-d6): δ 10.64(s, 0.30H), 10.60(s, 0.70H), 8.79(s, 0.30H), 7.91(s, 1.40H), 7.68(m, 0.30H), 7.48(m, 1.40H), 7.24(m, 0.90H), 6.90(m, 1.70H);13C NMR (DMSO): δ 168.9, 167.8, 143.5, 141.0, 136.6, 134.6, 133.70, 133.65, 130.7, 129.2, 125.4, 122.4, 121.6, 120.0, 112.3, 111.7, 109.9; Anal. Calcd for C15H9Br2NO2.0.2H2O: theory: C 44.79, H 2.36, N 3.48; found: C 44.94, H 2.12, N 3.33; HRMS: for C15H9Br2NO2[M-H]: theory: 393.8901, found: 393.8900. E2:1H NMR (DMSO-d6): δ 10.79(s, 0.75H), 10.75(s, 0.25H), 8.79(s, 1.50H), 7.94(s, 0.50H), 7.82(s, 0.75H), 7.76(d, 0.75H, J = 2Hz), 7.57(s, 0.25H), 7.46(d, 0.25H, J = 2Hz), 7.30(dd, 0.25H, J = 8.3Hz, 2Hz), 7.24(dd, 0.75H, J = 8.3Hz, 2Hz), 6.9(d, 0.25H, J = 8.3Hz), 6.84(d, 0.75H, J = 8.3Hz);13C NMR (DMSO-d6): δ 168.6, 167.5, 153.5, 152.7, 142.3, 139.7, 136.9, 136.3, 133.8, 130.1, 128.9, 127.3, 125.9, 125.4, 120.1, 112.3, 111.6, 111.3, 40.6, 40.4, 40.2, 40.0, 39.8, 39.6, 39.4; HRMS: C15H8ClBr2NO2[M-H]: theory: 425.8532, found: 427.8513. F2:1H NMR (DMSO-d6): δ 11.06(s, 0.6H), 11.03(s, 0.4H), 8.86(s, 1.2H), 8.30(d, 0.6H, J = 1.5Hz), 8.27(d, 0.4H, J = 1.5Hz), 7.99(s, 0.6H), 7.94(s, 0.6H), 7.88(m, 1H), 7.59(s, 0.4H), 6.99(d, 0.4H, J = 8.3Hz), 6.94(d, 0.6H, J = 8.1Hz), 3.85(s, 1.8H), 3.79(s, 1.2H);13C NMR (DMSO-d6): δ 169.2, 168.0, 166.8, 147.4, 144.9, 137.0, 136.5, 134.1, 130.9, 125.7, 123.0, 122.8, 121.1, 112.4, 111.8, 109.7, 52.4; HRMS: C17H11Br2NO4 [M-H]: theory: 451.8956, found: 451.8962. Bioevaluation. An initial growth inhibition assay was conducted using the MTS reagent to find the IC50and MTC values of each compound when co-incubated with L3.6pl pancreatic cancer cells for 24 and 72-hour timepoints. These values are listed in Table 2. ATTORNEY DOCKET NO.10669-426PC0 Table 2. Biological Evaluation of the GW5074 derivatives in L3.6pl human pancreatic cancer cells Compound Structure L3.6 pl IC50 (µM) MTC (µM) Tag 24 h 72 h 24 h 72 h B2 13.1 + / - 0.08 22.15+ / - 1.56 7.0 + / - 0.09 5.64 + / - 0.36 (GW5074) As shown in Table 2, the compounds demonstrated modest growth inhibition of L3.6pl cells in the 13-55 µM range after 24h or 72h incubation. In other embodiments, the invention extends these findings to now use PBT in combination with an immune checkpoint inhibitor. For example, the PD1 inhibitor had little effect on triple negative breast cancer cells, while PBT had a modest effect. However, the combination of DFMO, polyamine transport inhibitor like trimer44NMe and the PD1 inhibitor gave a dramatic response and even cures! Without being bound to a particular theory, it is believed that the PBT is priming the immune system and the PD1 inhibitor then unleashes the T cells to help clear the tumor cells. These outcomes have been observed in numerous cancers including melanoma, triple negative breast cancers and pancreatic cancers suggesting that this is a general immunotherapy that could be useful in many cancers beyond those investigated. Since ATTORNEY DOCKET NO.10669-426PC0 polyamines are regulated by Myc, the findings herein suggest that Myc driven tumors may be especially sensitive to this approach. Example 3 PBT and Immune Checkpoint Inhibitors for Treating Triple Negative Breast Cancer Using a mouse model of metastatic triple negative breast cancer, EO771.LMB tumor cells orthotopically injected into the mammary fat pad of syngeneic C57Bl / 6 mice to evaluate to what extent PBT (trimer44NMe PTI and DFMO) can resensitize tumors that are resistant to PD-1 immune checkpoint blockade (ICB) alone. The treatment scheme in Figure 13 shows that the mice were treated 3 times with anti-PD-1 monoclonal antibody (mAb) or a control isotype mAb and then daily with PBT for three weeks. There was no significant difference in tumor growth in mice treated with PBT alone, anti-PD-1 mAb alone, and vehicle control treated mice. However, tumor growth was significantly decreased in mice treated with PBT + anti-PD-1 mAb, and tumors in half the mice (3 / 6 mice) completely regressed and remained regressed for three weeks when treatment was stopped. The surviving mice were then challenged with completely regressed tumors with an additional orthotopic injection of EO771.LMB tumor cells in an opposite mammary fat pad and observed no secondary tumor growth for 2 additional months with PBT and anti-PD-1 mAb treatment still discontinued. This experiment demonstrated that PBT treatment can resensitize an ICB-resistant tumor leading to tumor regression and a durable anti-tumor immune memory as shown by no secondary tumor growth without additional treatment. These “cured” mice offer hope that PBT may also enhance anti-tumor efficacy of immune checkpoint inhibitors in patients with aggressive triple negative breast cancer, resulting in a long-term immune memory to prevent tumor recurrence and to improve patient survival. See Figure 19 Synergy. Figure 20 is a graph that was produced after running a linear mixed effects model with an interaction between time and the treatment groups (time*groups). The predicted log tumor volumes were then graphed at each time point. The p < .0001 indicates that the interaction is significant, meaning that there is evidence of a synergistic effect. Figure 21 provides evidence of synergy using a Bliss Combination Index (CI) that shows how the combination of PD-1 + PBT compares to what one would expect if the drugs simply added up (no interaction effect). The dotted line at 1.0 is the cutoff between synergy and antagonistic or additive effects. The Bliss Synergy Score (i.e. Bliss independence) in Figure 22 shows how much better the combination therapy works than one would expect if both drugs were working independently. A score greater than zero shows synergy. At each time point, the combination therapy shows a synergistic effect for both CI and Synergy Score. The HSA Combination Index (CI) of ATTORNEY DOCKET NO.10669-426PC0 Figure 22 shows how the combination of PD-1 + PBT compares to the most effective single treatment (either PD-1 or PBT). The dotted line at 1.0 represents an additive effect — meaning the combination is no better than the better monotherapy. Values below 1.0 indicate synergy, where the combination outperforms the best single agent. The HSA Synergy Score shows how much better the combination therapy performs compared to the individual drug. A score greater than zero indicates synergy; the higher the score, the stronger the synergy effect. Example 4: Inhibition of polyamine import is cytotoxic to chemo-resistant tumorspheres. ATP13A3 is an essential component of the polyamine transport system. ATP13A3 expression is elevated in tumors compared to normal tissue, and it is associated with poor overall survival rates in triple negative breast cancer (TNBC) and other cancer types (2,3). Data from cBioportal reveals that out of 10,851 patient samples, ATP13A3 is amplified in 4% of breast cancer of which 29.4% were HER2+, 6.3% ER+, and 56.3% TNBC. Recent experiments have explored to what extent intracellular polyamine accumulation is essential for survival of slow cycling, therapy-resistant TNBC tumor cells grown as tumorsphere cultures. Slow-cycling TNBC cells generated by acquired resistance to chemotherapy and / or immunotherapy share many properties of cancer stem-like cells, including their ability to metastasize readily and their ability to proliferate in low adherence cell culture conditions to generate floating tumorspheres (4-9). Disclosed herein is the discovery that ectopic ATP13A3 expression is highly elevated in slow-cycling (PKH26+label-retaining), chemotherapy-resistant TNBC tumorspheres (Figure 14). Briefly, human TNBC SUM159 cells were stained with PKH26, a red membrane dye that is diluted as cells proliferate, and grown as tumorspheres on ultralow adherence plates in serum-free medium. Viable PKH26 label-retaining (slow-cycling) tumorspheres following treatment with DOX were enriched for not only cell surface expression of ATP13A3 but also TNBC stem cell markers including CD304 (neuropilin), CD243 (i.e., multidrug resistant protein, MDR-1), RAC1B, and SETDB1, a histone methyltransferase amplified in TNBC that enhances stem-like properties and modulates the epithelial- mesenchymal transition (EMT) program (Figure 23B). Autophagic flux was also increased in DOX- resistant tumorspheres as measured by flow cytometry using the fluorescent dye Cyto-ID. Strikingly, co- treatment with polyamine transport inhibitors (PTI), the Trimer PTI or the arylpolyamine PTI, AP (10), increased the sensitivity of SUM159 tumorspheres to DOX (Figure 23A). These data suggest that TNBC therapy-resistance is dependent upon increased ectopic expression of ATP13A3 and that inhibition of polyamine import is cytotoxic to chemo-resistant tumorspheres. ATTORNEY DOCKET NO.10669-426PC0 CRISPR technology was used to create a functionally deficient (FD) ATP13A3 in EO771.LMB tumor cells to evaluate its role (and the role of polyamine transport) in tumor survival. EO771.lmb cells properly edited for ATP13A3FDalso express GFP and were sorted and expanded multiple times for GFP+to ensure a homogeneous population of EO771 cells that expressed functionally-dead ATP13A3. Compared to parental EO771 parental tumorspheres, mutant EO771-ATP13A3FDmutant tumorspheres were more sensitive to both doxorubicin and polyamine blocking therapy (PBT, DFMO + Trimer PTI) (Figure 24). Similarly, using tumorsphere cultures of L3.6pl pancreatic tumor cells with wildtype ATP13A3WTor with functionally dead ATP13A3FDprotein, it was found that the Trimer PTI is cytotoxic to gemcitabine-resistant L3.6pl tumorspheres with active ATP13A3 but not to gemcitabine-resistant L3.6pl-ATP13A3FDtumorspheres (Figure 25). Addition of spermidine partially rescued only the gemcitabine-resistant L3.6pl-ATP13A3WTtumorspheres co-treated with the PTI, suggesting that polyamine-dependency of chemo-resistant tumorspheres relies on a functional ATP13A3 to import polyamines. In addition, mutant EO771-ATP13A3FDtumor cells were less invasive (Figure 26) and less tumorigenic than parental EO771.lmb cells following orthotopic injection of the cells (1 x 105cells, 1:1 in Matrigel) in the mammary fat pad of syngeneic C57Bl / 6 mice (Figure 27). Not only did mutant EO771-ATP13A3FDtumors grow slower but they were more sensitive to treatment with DFMO alone (no PTI treatment) compared to that of parental EO771.lmb tumors with EO771-ATP13A3FDtumors totally regressing in 3 / 5 mice treated with DFMO alone (Figure 27). These experiments in this Example demonstrate that: ^ Functional ATP13A3 is important for TNBC tumor growth. ^ If tumor cell polyamine uptake via ATP13A3 is defective, then growth of tumors is more sensitive to DFMO alone. ^ Unlike parental EO771 tumors, complete tumor regression can be achieved in EO771ATP13A3-FDtumor cells with DFMO alone without treatment with the Trimer PTI (which targets ATP13A3). The extent to which genetic inhibition of ATP13A3 in EO771ATP13A3-FDtumor cells increases sensitivity to PD-1 blockade with or without treatment with DFMO was tested. It was found that a durable cure (long-term anti-tumor immune memory) can be achieved in mice treated with anti-PD-1 mAb + ATTORNEY DOCKET NO.10669-426PC0 DFMO if tumor cells have a functionally deficient ATP13A3, thus mimicking the synergistic anti-tumor effect observed with anti-PD-1 mAb + PBT (that inhibits ATP13A3 with its PTI component) (Figure 28). Example 5: PBT alleviates doxorubicin-promoted metastasis and decreases intratumor neutrophil extracellular trap (NET) formation in 4T1 tumors Following up on the data explained above, the following was also observed: 1) Trimer PTI alone can inhibit NETosis in in vitro experiments using murine bone marrow derived neutrophils as well as neutrophils isolated from human peripheral blood. Neutrophils were successfully isolated from plasma collected from healthy volunteers using a Polymorphprep gradient. The neutrophils were 98% CD16+by flow cytometry, had the typical segmented nuclei of mature neutrophils by microscopy, and underwent NET formation quickly following stimulation with platelet activated factor (PAF) (Figure 29). Using neutrophils isolated from the peripheral blood of healthy volunteers, it was also found that NETosis is stimulated following a 2 hour exposure to 12-O- tetradecanoylphorbol ester (TPA) and is inhibited with the addition of the Trimer PTI alone (Figure 30). Additional studies with isolated neutrophils have revealed that NETosis is further stimulated when neutrophils are exposed to serum from tumor-bearing mice but not with serum from non-tumor-bearing mice. Since NETosis can stimulate metastasis and tumor progression, the finding that in vitro NETosis can be inhibited with the Trimer PTI suggests that this polyamine blocking therapy may be useful in reducing metastasis and tumor progression in cancer patients with elevated levels of NETosis biomarkers. In addition, neutrophils were isolated from mouse bone marrow on a Percoll gradient and plated on poly-L-lysine–coated chamber slides for a 30-minute adherence step followed by addition of phorbol 12-myristate 13-acetate (PMA) to stimulate NETosis. It was found that treatment with the Trimer PTI inhibits PMA-induced NET formation using these murine bone marrow derived neutrophils. (Figure 31A, 31B). Further, it was discovered that cell surface expression of both ATP13A3 and citrullinated histone H3 (early NETosis marker) are increased after only 30 min of PMA stimulation and before the extrusion of DNA-histone NETs (Figure 31C). Altogether, these suggest a fascinating link between polyamine accumulation and NET formation and implicate ATP13A3 as a putative target for controlling NETosis- stimulated metastasis and therapy resistance. Example 6: PBT can protect against Doxorubicin-induced cardiotoxicity ATTORNEY DOCKET NO.10669-426PC0 Doxorubicin-induced cardiotoxicity is a serious, life-threatening side effect when treating cancer patients with this common chemotherapeutic agent. Doxorubicin, a potent anthracycline chemotherapy drug, is widely used to treat various cancers, according to the National Institutes of Health (NIH). However, its effectiveness is limited by a potentially severe side effect: cardiotoxicity, which can lead to irreversible heart damage and even congestive heart failure. Figure 32 shows that the combination of doxorubicin with PBT dramatically increases the survival rate in a mouse model. Thus, PBT is a suitable co-therapy to ameliorate the adverse effects of doxorubicin administration.

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P.; Ferguson, W.; Roberts, W.; Wada, R. K.; et al. A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed / Refractory Neuroblastoma. PLoS One 2015, 10 (5), e0127246. DOI: 10.1371 / journal.pone.0127246. (17) Alexander, E. T.; Fahey, E.; Phanstiel IV, O.; Gilmour, S. K. Loss of Anti-Tumor Efficacy by Polyamine Blocking Therapy in GCN2 Null Mice. Biomedicines 2023, 11 (10), 2703. DOI: 10.3390 / biomedicines11102703. (18) Alexander, E. T.; Mariner, K.; Donnelly, J.; Phanstiel IV, O.; Gilmour, S. K. Polyamine Blocking Therapy Decreases Survival of Tumor-Infiltrating Immunosuppressive Myeloid Cells and Enhances the Antitumor Efficacy of PD-1 Blockade. Mol Cancer Ther 2020, 19 (10), 2012-2022. DOI: 10.1158 / 1535- 7163.MCT-19-1116. (19) Alexander, E. T.; Minton, A.; Peters, M. C.; Phanstiel, O.; Gilmour, S. K. A novel polyamine blockade therapy activates an anti-tumor immune response. Oncotarget 2017, 8 (48), 84140-84152. DOI: 10.18632 / oncotarget.20493. (20) Casero, R. A., Jr.; Marton, L. J. Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases. Nat Rev Drug Discov 2007, 6 (5), 373-390. DOI: 10.1038 / nrd2243. (21) Gitto, S. B.; Pandey, V.; Oyer, J. L.; Copik, A. J.; Hogan, F. C.; Phanstiel, O.; Altomare, D. A. Difluoromethylornithine Combined with a Polyamine Transport Inhibitor Is Effective against Gemcitabine Resistant Pancreatic Cancer. Mol Pharm 2018, 15 (2), 369-376. DOI: DOI:10.1021 / acs.molpharmaceut.7b00718. (22) Mohammed, A.; Janakiram, N. B.; Madka, V.; Ritchie, R. L.; Brewer, M.; Biddick, L.; Patlolla, J. M.; Sadeghi, M.; Lightfoot, S.; Steele, V. E.; et al. Eflornithine (DFMO) Prevents Progression of Pancreatic Cancer by Modulating Ornithine Decarboxylase Signaling. Cancer Prev Res (Phila) 2014, in press. DOI: 10.1158 / 1940-6207.CAPR-14-0176. ATTORNEY DOCKET NO.10669-426PC0 (23) Quemener, V.; Moulinoux, J. P.; Havouis, R.; Seiler, N. Polyamine deprivation enhances antitumoral efficacy of chemotherapy. Anticancer Res 1992, 12 (5), 1447-1453. (24) Sekhar, V.; Andl, T.; Phanstiel IV, O. ATP13A3 facilitates polyamine transport in human pancreatic cancer cells. Sci Rep 2022, 12 (1), 4045. DOI: 10.1038 / s41598-022-07712-4. (25) Madan, M.; Patel, A.; Skruber, K.; Geerts, D.; Altomare, D. A.; Phanstiel, O. ATP13A3 and caveolin- 1 as potential biomarkers for difluoromethylornithine-based therapies in pancreatic cancers. Am J Cancer Res 2016, 6 (6), 1231-1252. References Cited for Examples 4-6: 1. Alexander ET, M. K., Donnelly, J, Phanstiel IV O, and Gilmour SK. (2020) Polyamine Blocking Therapy Decreases Survival of Tumor-Infiltrating Immunosuppressive Myeloid Cells and Enhances the Anti-Tumor Efficacy of PD-1 Blockade. Molecular Cancer Therapeutics 19, 2012-2022 2. Azfar, M., van Veen, S., Houdou, M., Hamouda, N. N., Eggermont, J., and Vangheluwe, P. (2022) P5B- ATPases in the mammalian polyamine transport system and their role in disease. Biochimica et biophysica acta. Molecular cell research 1869, 119354 3. Madan, M., Patel, A., Skruber, K., Geerts, D., Altomare, D. A., and Phanstiel IV, O. (2016) ATP13A3 and caveolin-1 as potential biomarkers for difluoromethylornithine-based therapies in pancreatic cancers. American journal of cancer research 6, 1231-1252 4. Basu, S., Dong, Y., Kumar, R., Jeter, C., and Tang, D. G. Slow-cycling (dormant) cancer cells in therapy resistance, cancer relapse and metastasis. Seminars in Cancer Biology, 2022, 78, 90-103. DOI: 10.1016 / j.semcancer.2021.04.021 5. Ehmsen, S., and Ditzel, H. J. (2021) Signaling pathways essential for triple-negative breast cancer stem- like cells. Stem cells (Dayton, Ohio) 39, 133-143 6. Liu, S., and Wicha, M. S. (2010) Targeting Breast Cancer Stem Cells. Journal of Clinical Oncology 28, 4006-4012 7. Wu, H. J., and Chu, P. Y. Epigenetic Regulation of Breast Cancer Stem Cells Contributing to Carcinogenesis and Therapeutic Implications. Int J Mol Sci 2021, 22, 8113. 8. Liu, S., Cong, Y., Wang, D., Sun, Y., Deng, L., Liu, Y., Martin-Trevino, R., Shang, L., McDermott, S. P., Landis, M. D., Hong, S., Adams, A., D'Angelo, R., Ginestier, C., Charafe-Jauffret, E., Clouthier, S. G., Birnbaum, D., Wong, S. T., Zhan, M., Chang, J. C., and Wicha, M. S. (2014) Breast cancer stem cells transition between epithelial and mesenchymal states reflective of their normal counterparts. Stem cell reports 2, 78-91 9. Crabtree, J. S., and Miele, L. Breast Cancer Stem Cells. Biomedicines 2018, 6, 77. 10. Muth, A., Kamel, J., Kaur, N., Shicora, A. C., Ayene, I. S., Gilmour, S. K., and Phanstiel IV, O. (2013) Development of polyamine transport ligands with improved metabolic stability and selectivity against specific human cancers. J Med Chem 56, 5819-5828

Claims

ATTORNEY DOCKET NO.10669-426PC0 CLAIMS What is claimed is:

1. Method for the manufacture of compound F2.

2. Method for the manufacture of compound C2.

3. Method for the manufacture of compound E2.

4. Use of F2 as a PTI in the treatment of proliferative diseases with cancers being preferred 5. Use of F2 in combination with a polyamine biosynthesis inhibitor (difluoromethylornithine, DFMO) for the treatment of proliferative diseases with cancers being preferred 6. Use of F2 in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of cancer 7. Use of F2 in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of cancer where F2 + DFMO provide an immunotherapy adjuvant which potentiates another immunotherapeutic agent such as a PD1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor or PD2 inhibitor with a PD1 inhibitor being preferred.

8. Use of F2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of cancer 9. Use of F2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of breast cancer 10. Use of F2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of triple negative breast cancer 11. Use of F2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of pancreatic cancer 12. Use of F2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of melanoma 13. Use of C2 as a PTI in the treatment of proliferative diseases like cancers 14. Use of C2 in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of proliferative diseases 15. Use of C2 in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of cancerATTORNEY DOCKET NO.10669-426PC0 16. Use of C2 in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of cancer where C2 + DFMO provide an immunotherapy adjuvant which potentiates another immunotherapeutic agent such as a PD1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor or PD2 inhibitor with a PD1 inhibitor being preferred.

17. Use of C2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of cancer 18. Use of C2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of breast cancer 19. Use of C2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of triple negative breast cancer 20. Use of C2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of pancreatic cancer 21. Use of C2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of melanoma 22. Use of E2 as a PTI in the treatment of proliferative diseases like cancers 23. Use of E2 in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of proliferative diseases 24. Use of E2 in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of cancer 25. Use of E2 in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of cancer where E2 + DFMO provide an immunotherapy adjuvant which potentiates another immunotherapeutic agent such as a PD1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor or PD2 inhibitor with a PD1 inhibitor being preferred.

26. Use of E2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of cancer 27. Use of E2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of breast cancer 28. Use of E2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of triple negative breast cancer 29. Use of E2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of pancreatic cancerATTORNEY DOCKET NO.10669-426PC0 30. Use of E2 in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of melanoma 31. Use of trimer44NMe or its pharmacologically acceptable salt as a PTI in the treatment of proliferative diseases such as cancer.

32. Use of trimer44NMe or its pharmacologically acceptable salt in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of proliferative diseases such as cancer.

33. Use of trimer44NMe or its pharmacologically acceptable salt in combination with a polyamine biosynthesis inhibitor (DFMO) for the treatment of cancer where trimer44NMe + DFMO provide an immunotherapy adjuvant which potentiates another immunotherapeutic agent such as a PD1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor or PD2 inhibitor with a PD1 inhibitor being preferred.

34. Use of trimer44NMe or its pharmacologically acceptable salt in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of cancer 35. Use of trimer44NMe or its pharmacologically acceptable salt in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of breast cancer 36. Use of trimer44NMe or its pharmacologically acceptable salt in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of triple negative breast cancer 37. Use of trimer44NMe or its pharmacologically acceptable salt in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of pancreatic cancer 38. Use of trimer44NMe or its pharmacologically acceptable salt in combination with a polyamine biosynthesis inhibitor (DFMO) and a PD-1 inhibitor for the treatment of melanoma 39. Combination therapy including a polyamine biosynthesis inhibitor and a polyamine transport inhibitor as an immune modulating therapeutic in the treatment of cancers with breast, colorectal, melanoma and pancreatic cancers.

40. Combination therapy including difluoromethylornithine (DFMO) and a polyamine transport inhibitor as an immune modulating therapeutic in the treatment of cancers with breast, colorectal, melanoma and pancreatic cancers being preferred.

41. Combination therapy including difluoromethylornithine (DFMO) and a polyamine transport inhibitor and a PD-1 inhibitor for use in the treatment of cancers with breast, colorectal, melanoma and pancreatic cancers being preferred.ATTORNEY DOCKET NO.10669-426PC0 42. Use of difluoromethylornithine and a polyamine transport inhibitor in combination with existing chemotherapeutic agents such as doxorubicin in the treatment of cancers.

43. Use of a PBT (polyamine transport inhibitor (e.g. trimer44NMe PTI) and a polyamine biosynthesis inhibitor (e.g.DFMO)) as an immune adjuvant agent to boost the efficacy of an immunotherapeutic agent which has shown insufficient of cancer when administered alone.

44. Use of PBT to metabolically reprogram immune cells to increase tumor sensitivity to an immunotherapeutic agent, wherein, optionally, the immunotherapeutic agent comprises at least one immune checkpoint inhibitor.

45. A method of treating cancer comprising co-administering PD-1 blockade and polyamine blocking therapy (PBT) in a synergistic amount, wherein the synergistic amount achieves a cancer cell killing effect at lower doses than what is achievable by administering PD-1 blockade or PBT alone.

46. A method for treating chemoresistant cancer exhibiting increased ectopic expression of ATP13A3, the method comprising administering a therapeutically effective amount of PBT, wherein the therapeutically effective amount inhibits ATP13A3 expression or activity.

47. A method comprising co-administering an adjunct cancer therapy protocol with PBT or PTI without DFMO at least one immune checkpoint inhibitor to achieve a durable anti-tumor immune response and immune memory in tumor that is being concurrently treated therewith but is resistant to immune checkpoint blockade.

48. A method for inhibiting NETosis and NET-stimulated tumor progression and metastasis, wherein the method comprises administering a therapeutically effective amount of a trimer PTI, either alone or in combination with a chemotherapeutic agent like doxorubicin.

49. A method comprising administering PBT to a subject in need, wherein the subject in need exhibits Doxorubicin-induced cardiotoxicity.

50. The use of any of the preceding claims, wherein use comprises administering a therapeutically effective amount of the named compound or compounds.

51. The therapy of any of the preceding combination therapy claims, where combination therapy comprises co-administering a therapeutically effective amount of each of the named compound or compounds to treat cancer.ATTORNEY DOCKET NO.10669-426PC0 52. A method of treating cancer comprising co-administering an immune checkpoint inhibitor and polyamine blocking therapy (PBT) in a synergistic amount, wherein the synergistic amount achieves a cancer cell killing effect at lower doses than what is achievable by administering an immune checkpoint inhibitor or PBT alone.

53. A method of treating cancer in a subject comprising co-administering a therapeutically effective amount of an immune checkpoint inhibitor and a therapeutically effective amount of a polyamine blocking therapy (PBT) to the subject.

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