A method for treating breast cancer by targeting nitric oxide pathway and PI3k pathwaya

Abstract

The present invention relates to a method for treating human breast cancers using combination therapy that includes therapeutically effective amounts of an iNOS inhibitor, a PI3K-ATK pathway inhibitor, and ataxane.

Description

Docket No. 38358.0004P1A METHOD FOR TREATING BREAST CANCER BY TARGETING NITRIC OXIDE PATHWAY AND PI3K PATHWAY1. FIELD OF THE INVENTION

[0001] Provided herein are methods for treating chemoresistant breast cancer and metaplastic breast cancer in a human subject in need thereof.2. BACKGROUND

[0002] Metaplastic breast cancer (MpBC) is a rare and highly aggressive malignancy that exhibits the most dismal prognosis of all breast cancers, with a survival rate of 8 months or less in patients with metastatic disease. MpBCs are typically triple-negative, lacking the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), and histologically characterized by the presence of both epithelial and mesenchymal components. Patients with MpBC receive similar therapeutic interventions to patients with non-metaplastic triple-negative breast cancers (non-MpTNBC); however, MpBC tumors are more chemoresistant and have a worse overall survival (OS) than non-MpTNBC. A need exists for new treatment modalities.3. SUMMARY OF THE INVENTION

[0003] The subject matter disclosed herein are methods for treating chemoresistant and / or metaplastic breast cancer in a human subject in need thereof with a treatment protocol that comprises administrations of an iNOS inhibitor (such as L-NMMA), a PI3K-ATK pathw ay inhibitor, and a taxane.

[0004] Embodiment 1 of the present disclosure is a method of treating chemoresistant and / or metaplastic breast cancer in a human subject in need thereof, said method comprising administering 15 to 25 mg / kg or 18 to 22 mg / kg or 20mg / kg of L-NMMA; administering a therapeutically effective amount of a taxane; and administering a therapeutically effective amount of a PI3K inhibitor.

[0005] Embodiment 2: The method of Embodiment 1, wherein the PI3K inhibitor is alpelisib, inavolisib, buparlisib, tasehsib, idelalisib, duvelisib. leniohsib, or copanlisib.- 1 -35416367. v7Docket No. 38358.0004P1

[0006] Embodiment 3: The method of Embodiment 1 or 2, wherein the PI3K inhibitor is alpelisib and optionally, the therapeutically effective amount is 100 mg to 150 mg or 150 mg to 200 mg or 150 mg to 225 mg or 200 mg to 250 mg or 250 mg to 400 mg or 300 mg to 375 mg or 350 mg or preferably, 200 mg or wherein the PI3K inhibitor is inavolisib and optionally, the therapeutically effective amount is 3 mg to 9 mg or 3 mg or 6mg or 9 mg.

[0007] Embodiment 4: The method of any one of Embodiments 1 to 3, wherein the PI3K inhibitor is administered every day of a treatment cycle.

[0008] Embodiment 5 : The method of any foregoing Embodiment, wherein the PI3K inhibitor is administered orally.

[0009] Embodiment 6: A method of treating chemoresistant and / or metaplastic breast cancer in a human subject in need thereof, said method comprising: administering 15 to 25 mg / kg or 18 to 22 mg / kg or 20mg / kg of L-NMMA; administering a therapeutically effective amount of a taxane; and administering a therapeutically effective amount of an mTOR inhibitor.

[0010] Embodiment 7: The method of Embodiment 6, wherein the mTOR inhibitor is an ATP-competitive mTOR kinase inhibitor.

[0011] Embodiment 8: The method of Embodiment 7, wherein the mTOR inhibitor is torin-1, tonn-2. vistusertib. AZD8055, MLN0128. PI-103, GDC-0980, INK126, WYE-354, WAY- 600, and WYE-687, Ku-0063794, XL388. PP242, PP244, or PP30.

[0012] Embodiment 9: The method of Embodiment 6, wherein the mTOR inhibitor is rapamycin, everolimus, umirolumus, zotarolimus, deforolimus, sirolimus, or temsirolumus.

[0013] Embodiment 10: A method of treating chemoresistant and / or metaplastic breast cancer in a human subject in need thereof, said method comprising: administering 15 to 25 mg / kg or 18 to 22 mg / kg or 20mg / kg of L-NMMA; administering a therapeutically effective amount of a taxane; and administering a therapeutically effective amount of an AKT inhibitor.

[0014] Embodiment 11 : The method of Embodiment 10, wherein the AKT inhibitor is capivasertib. perifosine, MK-2206, uprosertib, ipatasertib, afuresertib, or miransertib.

[0015] Embodiment 12: The method of any foregoing Embodiment, wherein L-NMMA is administered on days 1, 2, 3, 4, and 5 of a treatment cycle or at least 3, 4, or 5 days of the first 7 days of a treatment cycle.

[0016] Embodiment 13: The method of any foregoing Embodiment, wherein the taxane is docetaxel, paclitaxel, nab-paclitaxeL larotaxel, or cabazitaxel.Docket No. 38358.0004P1

[0017] Embodiment 14: The method of Embodiment 13, wherein the taxane is nab-paclitaxel and the therapeutically effective amount is between 240 mg / m2to 280 mg / m2or 260 mg / m2.

[0018] Embodiment 15: The method of Embodiment 14, wherein nab-paclitaxel is administered on days 1, 8, and / or 15 of a treatment cycle or administered once per week during a treatment cycle.

[0019] Embodiment 16: The method of Embodiment 13, wherein the taxane is docetaxel and the therapeutically effective amount is 90 to 110 mg / m2or 100 mg / m2once per treatment cycle.

[0020] Embodiment 17: The method of any one of the foregoing Embodiments, wherein is L- NMMA and the taxane are administered intravenously.

[0021] Embodiment 18: A method of treating chemoresistant and / or metaplastic breast cancer in a human subject in need thereof, said method comprising: administering 20 mg / kg of L-NMMA on days 1, 2, 3, 4, and 5 of a treatment cycle or at least 3, 4. or 5 days of the first 7 days of a treatment cycle; administering between 260 mg / m2of nab-paclitaxel on day 1, 8, and 15 of a treatment cycle or day 1 of a treatment cycle or administered once per week during a treatment cycle; and administering between 100 mg to 150 mg or 150 mg to 200 mg or 175 mg to 250 mg or 250 mg to 400 mg or 300 to 375 mg or 350 mg or 200 mg per day of alpelisib.

[0022] Embodiment 19: The method of Embodiment 18, wherein the alpelisib is administered every' day of a treatment cycle.

[0023] Embodiment 20: The method of Embodiment 18 or 19, wherein is L-NMMA and the nab-paclitaxel are administered intravenously.

[0024] Embodiment 21 : The method of Embodiment 18, 19, or 20, wherein the alpelisib is administered orally.

[0025] Embodiment 22: A method of treating chemoresistant and / or metaplastic breast cancer in a human subject in need thereof, said method comprising: administering 20 mg / kg of L-NMMA on days 1, 2. 3, 4, and 5 of a treatment cycle or at least 3. 4. or 5 days of the first 7 days of a treatment cycle; administering between 260 mg / m2of nab-paclitaxel on day 1, 8, and 15 of a treatment cycle or day 1 of a treatment cycle or administered once per week during a treatment cycle; and administering between 3 mg to 9 mg or 3 mg or 6mg or 9 mg of inavolisib per day.

[0026] Embodiment 23: The method of Embodiment 33, wherein the inavolisib is administered every' day of a treatment cycle.Docket No. 38358.0004P1

[0027] Embodiment 24: The method of Embodiment 22 or 23, wherein is L-NMMA and the nab-paclitaxel are administered intravenously.

[0028] Embodiment 25 : The method of Embodiment 22, 23 or 24, wherein the inavolisib is administered orally.

[0029] Embodiment 26: The method of any of the foregoing Embodiment, wherein the subject has a co-morbidity.

[0030] Embodiment 27 : The method of Embodiment 26, wherein the co-morbidity is obesity' (BMI>30 or >30% body fat for females or >25% body fat for males).

[0031] Embodiment 28: The method of any of the foregoing Embodiment, wherein the breast cancer is further characterized as HER2 negative metaplastic breast cancer (MpBC) or locally advanced MpBC.

[0032] Embodiment 29: The method of any of the foregoing Embodiment, wherein the breast cancer is further characterized as chemorefractory, locally advanced breast cancer (LABC), triple negative breast cancer (TNBC),

[0033] Embodiment30: The method of the foregoing Embodiment, wherein the chemoresistant breast cancer is further characterized as: HER2 negative MpBC with squamous and / or sarcomatoid elements, including osseous, chondroid, or spindle morphology; TNBC with squamous and / or sarcomatoid elements, including osseous, chondroid, or spindle morphology; or locally advanced inoperable or metastatic MpBC with measurable disease.

[0034] Embodiment 31 : The method of any of the foregoing Embodiment, wherein the subject may have received prior immunotherapy.

[0035] Embodiment 32: The method of any of the foregoing Embodiment, wherein the treatment cycle is between 14-30 days or 17 to 25 days or 20 to 23 days or 21 days.

[0036] Embodiment 33: The method of any of the foregoing Embodiment, wherein the subject completes 3, 4. 5, 6, 7, 8, 9, or 10 treatment cycles.

[0037] Embodiment 34: The method of any of the foregoing Embodiment, further comprising administering amlodipine at 10 mg orally once daily for 6 days each cycle or days 0 to 5 each cycle or at an amount and frequency effective to reduce risk of hypertension related to L- NMMA administration.

[0038] Embodiment 35: The method of any of the foregoing Embodiment, further comprising administering aspirin at an amount effective to 81 mg once daily.Docket No. 38358.0004P1

[0039] Embodiment 36: The method of any of the foregoing claims, further comprising administering metformin starting one week prior to a first treatment cycle at 500 mg to 2000 mg per day.

[0040] Embodiment 37: The method of any of the foregoing Embodiment, further comprising administering an effective amount of an antihistamine, such as cetirizine, daily as a prophylaxis for a skin rash; wherein, if cetirizine, the effective amount is at 10 mg per day.

[0041] Embodiment 37: The method of any of the foregoing Embodiment, wherein the breast cancer is metaplastic. The method of any of the foregoing claims, wherein the breast cancer is metaplastic.

[0042] Embodiment 38: The method of any of the foregoing Embodiment, wherein the breast cancer is chemoresistant or wherein the patient has previously received a therapy comprising a chemotherapeutic, optionally wherein the chemotherapeutic is a taxane.

[0043] Embodiment 39: The method of any of the foregoing Embodiment, wherein the subject has previously received an immunotherapy.4. BRIEF DESCRIPTION OF THE FIGURES

[0044] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0045] FIG. 1 shows clinical activity of L-NMMA combined with docetaxel in chemorefractory MpBC patients. (A) Waterfall Plot showing change in target lesion / tumor volume, best response (RECIST), and baseline iNOS H-score expression status in the 13 patients with available data from post-baseline assessments. PD - Progressive Disease, SD - Stable Disease. PR - Partial Response, and CR - Complete Response. iNOS H-Score Metric (0-50: None, 51-100: Low, 101-150: Moderate, >150: High) (B) Representative mammogram images at baseline (BL) and end of cycle 6 for Patient 100-048 with CR, showing shrinkage in target lesion size. (C) Hematoxylin and Eosin staining of BL tumor from same patient, Patient 100-048, showing metaplastic squamous differentiation and end-of-treatment (EOT) tissue showing residual keratinized and fibrotic tissue. Scale bars represent 100 pM. Representative images of n=3 tissue samples. (D) BL IHC staining of iNOS, PTEN, and phospho-Akt of tumor tissue from Patient 100-048 and associated EOT iNOS, pAkt, and PTEN staining in residual tissue. Scale bars represent 100 pM. (E) iNOS H-score for BL and EOT tumors in indicated responders and non-responders. BL and EOT H-scores are indicatedDocket No. 38358.0004P1 by red and blue box and whisker plots, respectively. R2-R4 indicate responder patients, and NR1-NR6 indicate non-responder patients. H-score analysis of 4-6 images per slide captured from tissues to cover entire tissue bed. The center bar indicates median, bounds of box represent lower and upper quartiles, and the whiskers indicate minimum and maximum of the dataset for each group. Statistical analysis by two-sided Student’s t test. (F) NOS2 alteration frequencies in various cancers from cBioPortal Combined Study Dataset (n=l 79290). Red symbolizes amplification, green - mutation, blue-deep deletion, and purple-structural variant. MpBC is about half red and green. (G) NOS2 normalized gene expression in MpBC / metaplastic-like TNBC (n=41. blue dots) and non-MpTNBC (n=137, red dots) from the ARTEMIS dataset. Dot and error bars represent mean ± SD. Statistical analysis by two- sided Student’s t test. (H) Kaplan-Meier metastasis-free survival analysis of MpBC and non- MpTNBC tumors based on expression status [high (red line) / low (blue line)] of NOS2 from ARTEMIS dataset. (I) Gene set enrichment analysis of the top represented upregulated hallmark gene sets based on normalized enrichment score (NES) from RNA-sequencing data from TCGA in human MpBC tumors (n=14) compared to invasive ductal carcinoma tumors (n=814).

[0046] FIG. 2 shows (A) Swimmer's plot summarizing all the patients in the study. Each bar represents 1 subject in the study. Bar length represents time on study. PD - Progressive Disease, SD - Stable Disease, PR - Partial Response, and CR - Complete Response, AE- Adverse Event, LABC-Locally Advanced Breast Cancer, MBC-Metastatic Breast Cancer (B) Waterfall plot with associated TP53 / PIK3CA / PIK3R1 AKT1 mutation status in 13 patients with available data from post baseline assessment. Blue bars in waterfall plot indicate metastatic breast cancer (MBC) and red bars (the two right most bars with a PR and CR) indicate locally advanced breast cancer (LABC). Black boxes below waterfall plot indicate mutation present, white boxes indicate wild-ty pe / proficient, grey boxes indicate data not available. (C) Gene set enrichment analysis of the top represented downregulated hallmark gene sets based on normalized enrichment score (NES) from RNA-sequencing data collected from TCGA in human MpBC tumors (n=14) compared to invasive ductal carcinoma (IDC) tumors (n=814).

[0047] FIG. 3 shows co-expression of iNOS and phospho- Akt is predominant in MpBC. (A) Network of protein interactions with iNOS generated by STRING analysis. Each network node represents one gene. Red line highlight interaction between NOS2 and AKT1. (B) Mutual Exclusivity Analysis of cBioPortal Combined Dataset of all cancers (n=l 79290)Docket No. 38358.0004P1 demonstrated a significant tendency toward co-occurrence for NOS 2 with PIK3CA. AKT1, PTEN. and RPL39 genomic alterations. mRNA expression and protein / phosphoprotein data were selected in analysis. Statistical analysis using one-sided Fisher’s Exact Test. (C) Immunoblotting analysis of iNOS. PTEN, phospho-Akt (Thr308 and Ser473) in a panel of breast cancer cell lines with known PIK3CA / PIK3R1 and PTEN mutation status. HSP90 was used as a loading control. Human mammary epithelial (HMEC) cell line (MCF-10A), ER+ breast cancer (ZR-7530, MCF-7, and T47D), HER2+ breast cancer (SKBR3, JIMT1, HCC1954), non-metaplastic TNBC (MDA-MB-231, MDA-MB-468, HCC1937, HCCC38, BT20). and metaplastic TNBC (HCC1806, BT549, Gs578T, SUMI 59) cell lines are indicated in black, red, blue, green, and purple, respectively. Blots shown are representative images of n=3 biological replicates. (D) Droplet digital PCR analysis of RPL39 A14V and PIK3CA hotspot mutations (E542, E545K, E41047L, II 1047R) and iNOS / phospho-Akt (Ser473) immunohistochemical expression status in PDXs of TNBC (n=12), ER+ (n=5). HER2+ (n=3), and MpBC (n=6). Green bars (G) represent PDX models that express the specifically indicated mutation in the column. Blue bars in right most column (depending on gradient) represent expression of phospho-Akt from low to high expression, a marker of PI3K / Akt activation. Orange bars in column next to right most column (depending on gradient) represent low to high iNOS expression. (E) Comparison of normalized iNOS and phospho-Akt (Thr308 and Ser473) protein levels among HMEC in comparison with MpBC, TNBC, ER+, and HER2+ breast cancer cell lines. Each dot represents a cell line showing a representative experiment, statistical analysis by two-sided Student’s t test comparing differences in protein expression ratios of MpBC to TNBC cell lines. Bars and error bars represent Mean ± SD. (F) Results of two-sided Fisher’s exact test comparing MpBC mutation status of RPL39 A14V and PIK3CA hotspot mutations in other breast cancer subtypes. (G) mRNA expression of NOS2 in all breast cancer PDX models. Values were compared to ACT value from PDX 4913 (TNBC) as a control that was set to 1 and represent the mean ± SD of three biological replicates. From left to right on the x-axis. Green bars are 2147 through MCI; Red bars are 0074 to SF-38A, Blue gars are 3963 to 3613B; and Purple bars are 4664 to PIM0 6.

[0048] FIG. 4 shows (A) Network of Protein interactions with iNOS, PI3K, EMT, and hypoxia-related genes generated by STRING analysis at high confidence. Each network node represents one gene. (B) Mutual Exclusivity Analysis of cBioPortal Combined Dataset of PanCancer Atlas (n=76639) and (C) Mutual Exclusivity Analysis of Breast Cancer StudiesDocket No. 38358.0004P1(n=8644) comparing genomic alteration co-occurrence / mutual exclusivity of NOS2 with PIK3CA, AKT1, PTEN. and RPL39 molecular alterations. mRNA expression and protein / phosphoprotein level data profiles were selected in analysis. Statistical analysis using one-sided Fisher’s Exact Test.

[0049] FIG. 5 shows Pan-NOSi L-NMMA enhances PI3Ki induced DNA damage in MpBC cell lines with PIK3CA / PIK3R1 mutations. (A) RPL39 and PI3K pathway gene alterations in MpBC cell lines. (B) Individual IC50 values of alpelisib in MpBC cell lines as determined by Cell Titer Gio Assay and IC50 values were determined using PRISM software. (C) Crystal violet staining of anchorage-dependent colony formation assay indicates the sensitivity of cells to DMSO, L-NMMA, alpelisib, or alpelisib combined with L-NMMA. Effect of treatments is shown for SUM159, Hs578T, BT549, and HCC1806 cell lines. (D) Quantification of caspase 3 / 7 activation over a 72-hour time course of 4 mM L-NMMA and 5 pM alpelisib. Data displayed are representative of 6-8 biological replicates and fold change of activation is compared to fluorescence values obtained from DMSO treatment arm. In the graphs, DMSO control is indicated in black, L-NMMA in red, alpelisib in blue, and L- NMMA+alpelisib in grey. (E) Nitrite / Nitrate values in SUM159 control and NOS2KO cells, as determined by colorimetric nitrite / nitrate assay kit (Sigma). (F) Densitometry analysis of immunoblots shown in FIG. 6D quantified using ImageLab Software (Biorad). Statistical analysis by Student’s / test. Analysis was performed from immunoblots of three independent experiments. (G-H) Immunofluorescence analysis of Rad51 foci (G) and yH2AX foci (H) formation. Images were captured at 100X magnification with confocal microscope. Scale bars represent 15 pM. Statistical analysis by Student’s t test. n=3. (I) Immunoblotting of yH2AX and Rad51 in SUM159 (PIK3CA mutated), Hs578T (PIK3R1 mutated), BT549 (PTEN deleted cell lines treated for 4-24 hours with DMSO control, 4 mM L-NMMA, 5 pM alpelisib, and L-NMMA combined with alpelisib. (J-K) dNTP quantification results from competitive PCR of BT549 (J) and SUMI 59 (K) cells treated for 8 hours with inhibitors. Statistical analysis by two-sided Student’s / test.

[0050] FIG. 6 shows pan-NOS inhibitor L-NMMA acts synergistically with isoform a- specific PI3K inhibitor alpelisib in MpBC cell lines with PIK3CA / PIK3R1 mutations. (A) Four MpBC cell lines were treated with dimethyl sulfoxide (DMSO), or increasing concentrations of L-NMMA, alpelisib, or combination for 72 hours. Cell growth was evaluated using Sulforhodamine B (SRB) assay. Sensitivity of MpBC cell lines to L-NMMA alone, alpelisib alone, or L-NMMA combined with alpelisib was compared to vehicle controlDocket No. 38358.0004P1 treated MpBC cells. Cell viability (left) and the combination index (right) are shown for each of these four cell lines and determined by CalcuSyn software. Fa, fraction affected. Bars and error bars represent mean ± SD of three biological replicates. (B) Protein levels of iNOS, phospho- Akt (Ser473 / Thr308), total Akt, and GAPDH in SUM159 control and different NOS2 knockout (NOS2KO) clones. N0S2K.0 clones were developed using iNOS Double Nickase CRISPR plasmids. Blots shown are representative images of n=2 biological replicates. (C) Cell Gio Titer Cell Viability Assay results of SUMI 59 control and N0S2K0 clones treated with alpelisib at varied concentrations for 72 hours. IC50 values were determined by GraphPad Prism software. N0S2K.0 #2 line drops to baseline first. N0S2K0 #3 line drops to baseline second, SUM159 Parenteral line drops to baseline third. (D) Immunoblotting of iNOS and PI3K signaling markers in SUMI 59 (PIK3CA mutated), Hs578T PIK3R1 mutated), BT549 (PTEN deleted) cell lines treated for 24 hours with DMSO control, 4 mM L-NMMA, 5 pM alpelisib, and L-NMMA combined with alpelisib. Blots shown are representative images of n=3 biological replicates. Densitometry quantification values were determined using ImageLab software (Biorad) and found in FIG. 5. (E) Immunoblotting of S -nitrosoglutathione reductase (GSNOR) and tubulin loading control of MpBC cell lines HCCI806, BT549, Hs578T, and SUM159 with densitometry analysis indicated in bar graph. Blots shown are representative images of n=2 biological replicates. (F) Extent of DNA damage, quantified by the comet tail moment in the neutral comet assay. Statistical analysis by two-sided Student’s t test. n=3 biological replicates per condition, 40 comets counted per biological replicate.

[0051] FIG. 7 shows NOS inhibition augments PI3K inhibitor and taxane treatment in vivo. (A) Schematics representing the MpBC PDX (BCM-3807, BCM-4664, PIM-010, and PIM- 084) experimental design. PDXs derived from human MpBCs were transplanted into cleared mammary fat pad of female NSG mice. When tumors reached 150-200 mm3, mice were randomized to receive vehicle control. NOS inhibition therapy (L-NMMA [400 mg / kg oral gavage on day 1. 200 mg / kg oral gavage on days 2-5 J + amlodipine [10 mg / kg intraperitoneal injection on days 1-5]), PI3K inhibitor alpelisib (35 mg / kg oral gavage on days 1-5), or the combination of both therapies as indicated. Caliper measurements were taken twice a week. Days in which mice were treated with therapies are indicated in red (first 5 days of a weekly cycle) and rest days are indicated in green (last 2 days of a weekly cycle). (B-E) Mean tumor volume and corresponding waterfall plots demonstrating maximal treatment response to single-agent or combination therapy in four MpBC PDX models ([BCM-3807, n=6], [PIM-Docket No. 38358.0004P1010, n=7], [PIM-084, n=5], and [BCM-4664, n=6]). Average tumor volume [0.5 xx (mm long dimension) xx (mm short dimension)2] and data points are mean tumor volume ± SEM. Statistical analysis for B-E by two-sided Student’s t test (*, p<0.05, **p<0.01). Each bar in waterfall is derived from the maximal response of a single tumor-bearing mouse to therapy. Lines and bars in the plots indicated in black represent vehicle control (topmost line), blue represent L-NMMA single-agent therapy, red represent alpelisib single-agent therapy, and grey (bottom most line) indicate dual-agent therapy. P-values: BCM-3807 (control vs L- NMMA+alpelisib \p=0.0217], L-NMMA vs L-NMMA+alpelisib \p=0.0398\, alpelisib vs L- NMMA+alpelisib [p= 456]), PIM-010 (control vs L-NMMA+alpelisib \p=0.006\. L- NMMA vs L-NMMA+alpelisib \p=0.0231}, alpelisib vs L-NMMA+alpelisib [p=0.0079]), PIM084 (control vs L-NMMA+alpelisib [p=0.023 ], alpelisib vs L-NMMA+alpelisib [p=0.016]), BCM-4664 (control vs L-NMMA+alpelisib [p=0.0032], L-NMMA vs L- NMMA+alpelisib p=0.0239], alpelisib vs L-NMMA+alpelisib \p=0.1275]). (F-G) Tumor volumes of BCM-4664 (F) and BCM-3807 (G) tumors treated with vehicle control (black, 1stfrom left graphs), docetaxel (purple, 2ndfrom left graphs), or combination therapy (docetaxel + NOS inhibition therapy [blue, 3rdfrom left graphs)], docetaxel + alpelisib [green, 4thfrom left graphs], and docetaxel + NOS inhibition therapy + alpelisib [red, 5thfrom left graphs]). When tumors reached 150-200 mm3, they were randomized into the respective treatment arms. Each graph line represents a replicate / treatment arm. (H-I) Kaplan-Meier Survival curves of model BCM-4664 (H) and BCM-3807 (I) treated with vehicle control, docetaxel, or combination therapy (dual / triple combination). An event was scored when a tumor reached 1,200 mm3or from death. Statistical analysis using Log-rank (Mantel-Cox) test (* p<0.05 and ***p< 0.001). The triple combination (red) has the longest survival curve in H and is trending to be the longest in I.

[0052] FIG. 8 shows (A-B) Body weights of mice implanted with BCM-4664 (A) and BCM03807 (B) tumors during treatment with inhibitors. (C) Representative images of immunohistochemistry (IHC) for iNOS and pAkt in single agent versus combined agent treated MpBC PDX tissues. Scale bars represent 100 pM. (D) H-score quantification results of iNOS and (E) pAkt (Ser473) IHC staining from tumor tissues. H-scores were evaluated in ten image fields per tissue sample (n=3 / treatment arm). Statistical analysis by two-sided Student’s T test. (F) Images of immunofluorescence stain of cleaved caspase 3 and DAPI nuclear stain in single-agent versus combined agent treated MpBC PDX tissues. Scale bars represent 100 pM. (G) Schematic representation of MpBC PDXs (BCM-3807 [PIK3CADocket No. 38358.0004P1 mutant] and BCM-4664 [PIK3CA WT]) experimental design. PDXs derived from human MpBC were transplanted into cleared mamman fat pad of female NSG mice. When tumors reached 150-200 mm3, mice were randomized to receive vehicle control, docetaxel (20 mg / kg on day 1), NOS inhibition therapy (L-NMMA [400 mg / kg oral gavage on day 1, 200 mg / kg oral gavage on days 2-5 J + amlodipine [10 mg / kg intraperitoneal injection on days 1 -5] ). PI3K inhibitor alpelisib (35 mg / kg oral gavage on days 1-5), or the combination of therapies as indicated. Caliper measurements were taken twice a week. (H) Cell Gio Titer Cell Viability Assay results of SUMI 59 control and NOS2KO clones treated with docetaxel at varied concentrations for 72 hours. IC50 values were determined by GraphPad Prism software. Clone #5 trends toward baseline first, followed by Clone #6.

[0053] FIG. 9 shows NOS inhibition induces epitheli al -to-mesenchymal transition reversal in MpBC. (A)Top GSEA pathways by normalized enrichment score (NES) from the Hallmark and Reactome collections, enriched in control (blue) and L-NMMA+alpelisib (red) treated PDX tumors. Light blue bars indicate non-significant pathways. (B) Representative immunofluorescence images of BCM-3807 tumors evaluated for (C) E-cadherin and Zebl protein expression. n=3 biological replicates per condition. Five images at lOx magnification per biological replicate covering the complete tissue bed were utilized for analysis. Scale bars represent 200 pM. Black, blue, red, and grey bars represent vehicle control, L-NMMA, alpelisib, and L-NMMA+alpelisib, respectively. (D) Immunoblotting of EMT markers in Hs578T (PIK3R1 mutated) cell lines treated with DMSO control, L-NMMA, alpelisib, and L-NMMA+alpelisib for 4-24 hours. (E) Morphology of SUM159 control cells and NOS2KO clone cells. 20x magnification and scale bars represent 200 pM. (G) Volcano Plot representing global transcriptional changes comparing SUM159 control cells and NOS2KO clone cells. Each data point represents a gene regulated by AP-1 transcription factor family. Differentially expressed genes (p<0.05) with a log2 fold change greater than 1 are upregulated genes (red dots), and less than -1 are down-regulated genes (green dots). Statistical analysis was performed using the Wald test. (H) Significantly differentially expressed genes clustered by their gene ontology (GO) with an adjusted P-value <0.05, tested using two-sided Fisher exact test (GeneSCF vl. l-p2). mRNA expression of TGFB1 (I) and LCN2 (J) from Parental SUM159 cells and NOS2KO clone cells and TGFB1 (L) and LCN2 (M) in SUMI 59 treated with non-targeting control siRNA and siRNAs specific to XBP1, CREB3, FOS, and JUN. Immunoblots of (F) EMT and iNOS associated proteins, (K) LCN2, TGF0 [active form], (N) phospho-cJun (Ser63 / Ser73), (O) S-nitrosylation of JNK (SNO-Docket No. 38358.0004P1JNK), and HSP90 loading control in Parental SUM159 cells and NOS2KO clone cells. For C, I, J, and K-O, Statistical analysis by two-sided Student’s t test. Bars and error bars represent the mean ± SD of three biological replicates. For all Blots, images shown are representative of n=3 biological replicates, and graph represents SNO-JNK / JNK protein expression ratios from n=3 biological replicates.

[0054] FIG. 10 shows (A) GSEA plots of keratinization, formation of the cornified envelope, and EMT pathways. NES, nominal p value, and false discovery’ rate (FDR) are indicated. (B) Representative immunofluorescence images of PIM-010 tumors evaluated for (C) E-cadherin and Zebl protein expression. Statistical analysis by Student’s t test. n=3. Images captured at lOx magnification at scale bars represent 200 pM. (D) Immunoblotting of EMT markers in SUMI 59 (PIK3CA mutated) cell lines treated for 4-24 hours with DMSO control, L-NMMA, alpelisib, and L-NMMA combined with alpelisib. (E) Migration of Parental SUMI 59 and NOS2KO cells following treatment with vehicle (DMSO) or different concentrations of alpelisib for 24 hours. Scale bars represent 500 pM. (F) Graph indicates the mean of migrated cells per field ± SD of three independent experiments; two-tailed Student t test. (G) Representative images of cell morphology' of Parental SUMI 59 and NOS2KO clone cells treated with TGF[3 and (H) corresponding immunoblots of mesenchymal transcription factors and Vimentin from untreated / TGF[3-treated cells. 20x magnification and scale bars represent 200 pM. (I) mRNA expression of XBP1, CREB3, FOS, and JUN from qPCR analysis of Parental SUM159 cells and cells treated with non-targeting control siRNA and gene-specific siRNAs. Statistical analysis by Student’s t test. n=3 replicates. (J and K) Immunoblots of (J) LCN2, TGF0 [active form], (K) phospho-cJun (Ser63 / Ser73) and HSP90 loading control in Parental BT549 cells and NOS2KO clone cells. (L) Morphology of BT549 control and NOS2KO clone cells. 20x magnification and scale bars represent 200 pM. (M) Immunoblots of EMT and iNOS associated proteins, (N) phospho-cJun (Ser63 / Ser73) and HSP90 loading control, and (O) LCN2, TGF[3 [active form], and HSP90 loading control in Hs578T cells, and Hs578T cells treated with non-targeting control, and siRNAs specific to NOS2 for 96 hours. Bars and error bars represent the mean ± SD of three biological replicates. For all Blots, images shown are representative of n=3 biological replicates.

[0055] FIG. 11 shows L-NMMA and alpelisib combined with taxane chemotherapy is effective at targeting breast cancer stem cells. (A) Schematic showing experimental outline to test the tumor initiating and self-renewal capacity of MpBC PDX tumor model BCM-3807 after treatment with targeted therapies with or without taxane chemotherapy. Figure createdDocket No. 38358.0004P1 wi th Biorender.com (B) Primary and (C) Secondary7MSFE % values of tumors from each indicated treatment group after 14 days of treatment. Tumors were treated in vivo for 14 days, collected, dissociated into single cell suspensions, and subsequently plated under mammosphere conditions (60,000 cells per well in 6-well low adherent plates in supplemented MammoCult Media) To determine secondary MSFE, primary mammosphere were collected, dissociated, and replated under mammosphere conditions. A two-tailed Student t test was conducted to evaluate p values comparing each treatment condition to vehicle control. Bars and error bars represent the mean ± SD of four biological replicates / mice per condition. P-values indicated in red compare triple combination therapy to both double combination therapies. P-values indicated in black compare indicated treatment arm to vehicle control. (D and E) CD44+ / CD24- (BCSC marker) results (flow cytometry) from tumors after treatment. A two-tailed Student t test was conducted to evaluate p values comparing each treatment condition to vehicle control. Bars and error bars represent the mean ± SD of four biological replicates / mice per condition. (F) Limiting Dilution Assay: BCM- 3807 tumors from docetaxel and triple combination (docetaxel+L-NMMA+alpelisib) treated mice (14 days) were dissociated and pooled. A total of 1000, 10000, and 100000 cells from each group were transplanted into the mammary gland fat-pad of 4- to 6-week-old mice (n=13-15 mice / group). Tumor incidence was reported at 12 weeks post-transplantation. Stem cell frequency fractions and overall statistical analysis using Chi-squared test comparing differences in stem cell frequencies between treatment groups was evaluated using available ELDA software, see Hu, Y, and Smyth, GK (2009). ELDA: Extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. Journal of Immunological Methods 347. 70-78; software accessible at bioinf.wehi.edu.au / software / elda / . (G-H) Flow cy tometry’ analysis of ALDH1+ cells in BCM-3807 tumors after 14 days of treatment with vehicle control, single-agent therapy (L- NMMA or alpelisib), and dual-agent therapy. Statistical analysis by two-sided Student’s t test. Bars and error bars represent the mean ± SD of five biological replicates / mice per condition.

[0056] FIG. 12 shows (A) Limiting Dilution Assay: PDX-3807 tumors from vehicle- and treated mice were dissociated and pooled. A total of 30,000 or 50,000 cells from each group were transplanted into the mammary gland fat-pad of 4- to 6-week-old mice. Tumor incidence was reported at 12 weeks post-transplantation. Data were analyzed by pairwise comparisons with Fischer exact test. (B-E) Representative immunofluorescence images (B)Docket No. 38358.0004P1 of BCM-3807 and (D) PIM-010 tumors evaluated for ALDH1 protein expression with (C and E) corrected total cell fluorescence. Statistical analysis by Student’s r test. n=3 mice per treatment arm. Images captured at lOx magnification and scale bars represent 200 pM.

[0057] FIG. 13 shows EMT reversal in MpBC human tumor biopsies after L- NMMA+taxane therapy. (A) Representative immunofluorescence images of tumor biopsy for patient 100-013 (responder) and (B) patient 100-009 (non-responder) stained for Zebl (red), E-cadherin (green), ALDH1 (green), and DAPI (blue). Images magnification lOx; scale bars represent 100 pM. Corrected total cell fluorescence analysis quantified using ImageJ software for (C) Zebl. (D) E-cadherin, (E) ALDH1 in patient tumor biopsies at baseline (BL) and end-of-treatment (EOT). Immunofluorescence analysis obtained from 5 images per slide captured from tissues to cover entire tissue bed. Statistical analysis by two-sided Student’s t test. Bars and error bars represent ±SD (F) Ratio of E-cadherin / Zeb expression (corrected total cell fluorescence) in patient tumor biopsies at BL and EOT. Statistical analysis was performed by Student’s t test. Red and blue bars indicate data for BL and EOT corrected total cell fluorescence, respectively. R2-R4 indicate responder patients, and NR1-NR6 indicate non-responder patients. (G) Schematic representing potential mechanism of action of NOS inhibition inducing EMT reversal. iNOS, inducible nitric oxide synthase; EMT, epithelial-to- mesenchymal transition; JNK. c-Jun N-terminal kinase; PI3K, phosphoinositide 3-kinase (PI3K); TGFB1, transforming growth factor beta 1; LCN2, lipocalin, AP-1, activator protein 1. Figure created with Biorender.com

[0058] FIG. 14 shows Representative images of Hematoxylin and Eosin staining of baseline tumor from responder 100-013 (A) and non-responder 100-009 (B) and end-of-treatment tissue. Light microscope images captured at 4x and 20x using a Nikon eclipse 90i microscope with a DS-Fil bright field camera (Nikon Instruments Inc). Scale bars for 4x and 20x image are 500 pM and 100 pM, respectively.

[0059] FIG. 15 shows (A) Example of gating strategy’ for flow cytometry’ analysis of breast cancer stem cells (CD44+ / CD24-) derived from metaplastic breast cancer patient derived xenograft tissues used in FIG. 1 ID. (B) Example of gating strategy for flow cytometry analysis of breast cancer stem cells (ALDH1+) derived from metaplastic breast cancer patient derived xenograft tissues used in FIG. 11G.Docket No. 38358.0004P15. DETAILED DESCRIPTION Definitions

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary' skill in the art to which this disclosure belongs. If there is a plurality of definitions for terms cited herein, those in this section prevail unless otherwise stated.

[0061] Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. As used in this specification and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise, e.g., "a compound" includes a plurality of compounds. Thus, for example, a reference to "a method" includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0062] The subject matter disclosed herein are methods for treating chemoresistant and / or metaplastic breast cancer in a human subject in need thereof with a treatment protocol that comprises administrations of an iNOS inhibitor (such as L-NMMA), a PI3K-ATK pathway inhibitor, and a taxane. A PI3K-ATK pathway inhibitor can be a PI3K inhibitor, ATK inhibitor, or an mTor inhibitor.Patient Populations

[0063] Metaplastic breast cancers are a heterogenous group of invasive breast cancers which share differentiation toward squamous or mesenchymal-appearing elements. In an embodiment of treating metaplastic breast cancer (MpBC), the breast cancer can be locally advanced metaplastic breast cancer or metastatic, metaplastic breast cancer, which optionally is HER2 negative. In some embodiments, the patient has triple-negative breast cancer (TNBC). TNBC are tumors that lack expression of estrogen receptor (ER) and progesterone receptor (PR), and lack of HER2 overexpression and / or HER2 gene amplification. In some embodiments, the metaplastic cancer can be squamous and / or sarcomatoid predominate.

[0064] The treatment embodiments described herein can be for both first- and second-line human subjects with breast cancer. In some embodiments, subjects may have received prior immunotherapy, as per standard of care. In some embodiments, the subjects did not respond or have ceased responding to the immunotherapy.Docket No. 38358.0004P1

[0065] In embodiments of treating chemoresistant breast cancer, the human subject has previously been refractory to standard of care, for example, the subjects did not respond or have ceased responding to a first line therapy. In embodiments, the breast cancer can be metaplastic, such as locally advanced metaplastic breast cancer, or metastatic, HER2 negative metaplastic breast cancer.

[0066] The treatment embodiments described herein can be for treating breast cancer that is HER2 negative MpBC with squamous and / or sarcomatoid elements, including osseous, chondroid, or spindle morphology; triple negative breast cancer (TNBC) with squamous and / or sarcomatoid elements, including osseous, chondroid, or spindle morphology’; or locally advanced inoperable or metastatic MpBC with measurable disease.

[0067] In embodiments, the human subject is positive for a PIK3CA-mutated or PIK3R1 mutated breast cancer. The breast cancer can be metaplastic, metastatic or locally advanced, HER2 negative or triple negative, or a combination thereof. In embodiments, the breast cancer is PIK3CA-or PIK3Rlmutated, locally advanced or metastatic MpBC, optionally HER2 negative. iNOS Inhibitors

[0068] Inducible nitric oxide synthase (iNOS) is a crucial enzyme involved in monocyte cell response towards inflammation, and it is responsible for the production of sustained amounts of nitric oxide. iNOS expression is increased and associated with poor prognosis in invasive TNBC and MpBC. It has been shown that nitric oxide (NO) through iNOS induces many of the major oncogenic pathways such as RAS / ERK. HIFla, NF-KB, and others making this a unique oncogenic driver. One of the major oncogenic pathways activated by iNOS / NO is the phosphatidylinositol 3-kinase (PI3K) / Akt pathway. Alterations in the PI3K / Akt pathway have also been linked with chemotherapy resistance, especially^ in MpBC.

[0069] Methods of treating chemoresistant and / or metaplastic breast cancer described herein involve administering an iNOS inhibitor. An embodiment of the iNOS inhibitor can be NG- monomethyl-l-arginine . also referred to as L-NMMA or tilarginine. Other embodiments of the iNOS inhibitor are L-NAME, 2-Iminobiotin, MTR 104, L-NILTA (prodrug of LNIL), GW274150, Ronopterin, S-Ethylisothiourea, 4-(ethanimidoylamino)-N-(4- fluorophenyljbenzamide hydrobromide (FAB 1020), or N-{3- [(ethanimidoylamino)methyl]benzyl}-l-prolinamidedihydrochloride (CM554).Docket No. 38358.0004P1

[0070] The iNOS inhibitor, such as any one of the foregoing examples, can be administered on days 1, 2, 3, 4, and 5 of a treatment cycle or at least 3, 4, or 5 days of the first 7 days of a treatment cycle.Taxanes

[0071] Methods of treating chemoresistant and / or metaplastic breast cancer descnbed herein involve administering a taxane. In an embodiment, the taxane used in the treatment is docetaxel. The method can comprise administering docetaxel at 60-100 mg / m2once per treatment cycle.

[0072] In an embodiment, the taxane used in the treatment is paclitaxel. The method can comprise administering paclitaxel at 135 mg / m2to 175 mg / m2intravenously once per treatment cycle.

[0073] In an embodiment, the taxane used in the treatment is nab-paclitaxel. The method can comprise administering nab-paclitaxel at 240 mg / m2to 280 mg / m2intravenously. In some embodiments, the method comprises administering nab-paclitaxel at 260 mg / m2intravenously. In embodiments, the nab-paclitaxel is administered on day 1, 8, and 15 of a treatment cycle or on day 1 of a treatment cycle or administered once per week during a treatment cycle.

[0074] In an embodiment, the taxane used in the treatment is larotaxel.

[0075] In an embodiment, the taxane used in the treatment is cabazitaxel. The method can comprise administering cabazitaxel at 20-25 mg / m2once per treatment cycle. In embodiments, the patient is further administered 10 mg of prednisone daily.PI3K-ATK Pathway Inhibitors

[0076] Methods of treating chemoresistant and / or metaplastic breast cancer described herein involve administering a therapeutically effective amount of a PI3K-ATK Pathway Inhibitor.

[0077] In an embodiment, the PI3K-ATK Pathway Inhibitor used in the described treatments is a therapeutically effective amount of a PI3K inhibitor, particularly PI3K-a inhibitor. An example of such inhibitor is alpelisib. A therapeutically effective amount of alpelisib is a daily dose of 100 mg to 400 mg, such as 100 mg to 200 mg or 150 mg to 250 mg or 250 mg to 400 mg or 300 mg to 375 mg. In some embodiments, a therapeutically effective amount of alpelisib is 170 mg to 230 mg or 200 mg daily. In the event of an adverse event, the alpelisib can be administered for 40%-60% or 45%-55% of the treatment cycle days, such as 10 days of a 21 -day treatment cycle. In an embodiment, the administration of alpelisib is on consecutive days until 40-60% of treatment cycle days is achieved.Docket No. 38358.0004P1

[0078] Another example of a PI3K-a inhibitor is inavolisib. A therapeutically effective amount of is 3 mg to 9 mg or 3 mg or 6mg or 9 mg per day. In an embodiment, the dose in an initial treatment cycle can by 9 mg per day. To manage inavolisib-caused adverse events, the dose of inavolisib be reduced from 9 mg per day to 6 mg per day and further reduced from 6 mg per day to 3 mg per day.

[0079] Other PI3K inhibitors suitable for use with the methods described herein include buparlisib, serabelisib, taselisib, gedatolisib, idelalisib, duvelisib, leniolisib, ZSTK474, and copanlisib.

[0080] In an embodiment, the PI3K-ATK Pathway Inhibitor used in the treatment is a therapeutically effective amount of an ATK inhibitor. Examples of such inhibitors are capivasertib, perifosine, MK-2206, uprosertib, ipatasertib, afuresertib, and miransertib. In an embodiment, the ATK inhibitor is capivasertib and the therapeutically effective amount is 200 mg to 400 mg twice daily for 4 days with 3 days off per week. An initial dose can be 400 mg twice daily for 4 days with 3 days off per week. In the event of an adverse event caused by capivasertib, the dose can be reduced from 400 mg to 320 mg twice daily for 4 days with 3 days off per week and further reduced from 320 mg to 200 mg twice daily for 4 days with 3 days off per week.

[0081] In an embodiment, the PI3K-ATK Pathway Inhibitor used in the treatment is an mTOR Inhibitor. In an embodiment, the mTOR inhibitor is an ATP-competitive mTOR kinase inhibitor, such as torin-1, torin-2, vistusertib, AZD8055, MLN0128, PI-103, GDC- 0980, INK126, WYE-354, WAY-600, WYE-687, Ku-0063794, XL388, PP242, PP244, and PP30. Other examples of mTOR inhibitors are rapamycin. everolimus, umirolumus, zotarolimus, deforolimus, sirolimus, and temsirolumus.

[0082] The PI3K-ATK Pathway Inhibitor, particularly any one of the foregoing, can be administered daily or even,' other day or 3-4 days per week with 4-3 days off per week. Treatment Cycles

[0083] A treatment cycle is between 14-30 days or 17 to 25 days or 20 to 23 days or 21 days. Supplemental Medicines

[0084] In an embodiment, the subjects are administered various medicines to mitigate or sen e as prophylaxis of side effects the agents used in the described combination therapy.

[0085] In addition to the three agents described above, the method can further comprise administering amlodipine at 10 mg orally once daily for 6 days each cycle or days 0 to 5 eachDocket No. 38358.0004P1 cycle or at an amount and frequency effective to reduce risk of hypertension related to administration of a iNOS inhibitor, such as L-NMMA, or a PI3K-ATK Pathway Inhibitor.

[0086] In addition to the three agents described above, the method can further comprise administering aspirin at an amount effective to 81 mg once daily.

[0087] In addition to the three agents described above, the method can further comprise administering metformin starting one week prior to a first treatment cycle at 500 mg to 2000 mg per day.

[0088] In addition to the three agents described above, the method can further comprise administering an effective amount of an antihistamine, such as cetirizine, daily as a prophylaxis for a skin rash; wherein, if cetirizine, the effective amount is at 10 mg per day.

[0089] To be sure, a method described herein can comprise administering the three agents described above as well as the agents described in this section, namely amlodipine and an antihistamine, and metformin, and optionally aspirin, and optionally at the amounts specified above.EXAMPLES Example 1: NOS inhibition sensitizes metaplastic breast cancer to PI3K inhibition and taxane therapy via c-JUN repression

[0090] NOS inhibition with the pan-NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) decreased tumor cell proliferation, mammosphere formation, and migration in vitro and reduced tumor development and growth as well as lung metastasis in TNBC patient-derived xenograft (PDX) models.

[0091] Furthermore, NO can uniquely activate multiple oncogenic signaling pathways including those mediated by PI3K, extracellular signal-regulated kinase (ERK), [3-catenin, transforming growth factor beta (TGFP) and hypoxia-inducible factor (HIF). Considering that MpBC produces high levels of NO that can activate PI3K signaling and harbors aberrations in the PI3K pathway, we assessed a combinatorial approach of co-targeting NOS and PI3K / AKT, using the FDA approved a-specific PI3K inhibitor alpelisib (Piqray®, Novartis) and pan-NOS inhibitor L-NMMA, as an effective therapeutic strategy.

[0092] Patient-derived MpBC cell lines and PDX models demonstrate that iNOS inhibition augments the efficacy of PI3K inhibitor therapy against MpBC. This combinatorial effect is mediated in part through NOS inhibition inducing a reversal of EMT programming in MpBCDocket No. 38358.0004P1 cells, leading to enhanced chemosensitivity. Combining NOS and PI3K inhibition is an effective therapeutic strategy to treat MpBC by reversing EMT and decreasing CSCs, rendering MpBC tumors more chemosensitive.

[0093] Materials and Methods

[0094] Cells and Reagents

[0095] Cell lines used in this study were obtained from ATCC, used within three passages since thawing, tested negative for Mycoplasma, and authenticated by short tandem repeat profiling, and cultured in media containing 10% FBS (GenDepot) and 1% antibiotic- antimycotic reagent (GenDepot). Media used for cell lines is indicated in Table 1 below. L- NMMA was obtained from the cGMP facility at Houston Methodist Research Institute, PI3K inhibitor alpelisib was purchased from Cayman Chemicals, and docetaxel was purchased from Houston Methodist Pharmacy. Cells were treated with inhibitors in serum-free media (1% FBS) for specific time points indicated in the results section.Table 1

[0096] For the CRISPR knockout of NOS2, cells were transfected with the NOS2 Double Nickase Plasmid (Santa Cruz Biotechnology’)- After puromycin selection, resistant clones were selected by immunoblotting for iNOS expression and nitrite / nitrate colorimetric assay (Sigma). For siRNA knockdown, cells were transfected in a 6-well-plate with ON- TARGETplus SMARTpool human CREB3, XBP1, FOS, and JUN siRNA pools or nonDocket No. 38358.0004P1 targeting siRNA pools as control (Horizon Discovery, Dharmacon) using Lipofectamine RNAiMax (ThermoFisher). For TGFP rescue experiments, SUM159 Parental and NOS2KO cells were treated with TGFP (5 ng / ml; Cell Signaling) for 24 hours.

[0097] For qPCR analysis, RNA was isolated from breast cancer cell lines and PDX models using Qiagen RNeasy Kit according to manufacturer’s instructions. cDNA synthesis was performed using Superscript IV VILO Master Mix (ThermoFisher) and qPCR was performed using PowerSYBR Green PCR Master Mix (Applied Biosystems). The ddPCR experiments were performed using a QX200 ddPCR system (BioRad) as described previously 8 and analyzed with QuantaSoft software. Information about cell lines, primers, and antibodies are provided in Tables 2-4 below.Table 2Table 3Docket No. 38358.0004P1Table 4

[0098] In Vitro Assays

[0099] For Cell Viability Assay, treated MpBC cells were analyzed with Sulforhodamine B assay following manufacturer’s instructions (Abeam) and Cell Titer Gio assay (Promega). For Caspase 3 / 7 assay, MpBC cells were treated with inhibitors at physiologically relevant concentrations for 72 hours (L-NMMA at 4 mM, alpelisib at 5 pM. or combination of two drugs), and then incubated with the caspase 3 / 7 reagent, following manufacturer’s instructions (Promega). MpBC cell lines (SUM159, Hs578T, HCC1806, and BT549) were used for drug combination assay. L-NMMA and alpelisib were each arrayed in 96-well plates and serially diluted two-fold, y ielding a concentration equal to IC50 of each drug in MpBC cell lines. Cells were seeded in 96-well plates at a concentration of 3.0 x 104 cells / mL in 100 pL of medium per well, and then treated with a single drug or with a combination of L-Docket No. 38358.0004P1NMMA and alpelisib for another 72 hours. When testing for synergy of the drug combination, L-NMMA and alpelisib were plated serially onto MpBC cells with constant ratio concentrations in 96-well plates with Eppendorf Xplorer plus Electronic Single Channel Pipette. Combination index (CI) and fraction affected (Fa) values were calculated using CalcuSyn software. GraphPad Prism 6 software was used to plot dose response and caspase 3 / 7 activation plots and determine IC50 concentration. For Anchorage-Dependent Colony Formation Assay, MpBC cells were plated and treated with inhibitors accordingly. After 72 hours of inhibitor treatment, cells were washed with ice cold PBS, fixed with ice cold 100% methanol, stained with 0.5% crystal violet solution, washed, and dried well images were captured. Neutral comet assays were performed using the CometAssay Kit (Trevigen), as per the manufacturer’s instructions. Comet tail area was measured using CaspLab software, and calculations were averaged from three independent experiments.

[0100] Rad51 and YH2AX foci immunofluorescence

[0101] Immunofluorescence was performed following standard protocols. 57 MpBC cells were treated for 24 hours with DMSO control, L-NMMA, alpelisib, or combination (L- NMMA+alpelisib) on chambered cell culture slides (Coming). Cells were fixed, blocked, incubated with primary / secondary antibodies, and mounted coverslips using Antifade Mounting Medium with DAPI (Vector Laboratories). Images were acquired with Nikon AIR confocal imaging system with NIS Elements software (Nikon). Rad51 and YH2AX foci were quantified in 100 cells per replicate using ImageJ software.

[0102] Nucleotide Quantification

[0103] MpBC cells were seeded at a density of 2x106 cells and treated with DMSO, L-NMMA, alpelisib, or L-NMMA+alpelisib for 8 hours. After treatment, cells were prepared for dNTP quantification.

[0104] Biotin-Switch Assay

[0105] A modified biotin-switch assay was performed. SNO-JNK was detected using an S-nitrosylated protein detection kit (Cayman Chemicals. #10006518) according to manufacturer’s instructions and all steps were carried out in the dark. Biotinylated proteins were then purified with NeutrAvidin Plus Ultralink Resin (ThermoFisher), separated by SDS- PAGE, and immunoblotting to detect JNK expression.

[0106] RNA-Sequencing and Gene Set Enrichment Analysis (GSEA)

[0107] RNA samples were extracted with Qiagen RNAeasy Kit following manufacturer’s instructions. RNA was sent out to commercial companies, Novogene andDocket No. 38358.0004P1Genewiz for library preparation, high throughput sequencing using Illumina sequencers, and analysis (gene ontology / GSEA and differential gene expression analysis, https: / / www.gsea- msigdb.org / gsea / index.jsp). ARTEMIS dataset (MD Anderson Cancer Center) was analyzed using R Survminer package for survival analysis. Kaplan-Meier method was used to draw survival curves, and the log-rank test was performed to evaluate survival difference.

[0108] Immunoblotting

[0109] Whole cell lysates were made in IX RIPA Buffer (Sigma) containing Protease and Phosphatase Inhibitor Cocktail Solutions (GenDepot). Samples were boiled in 4x Laemmli Sample Buffer (Biorad) and subjected to SDS-PAGE electrophoresis (Bio-Rad). Proteins were transferred onto activated PVDF membranes (Bio-Rad). Membranes were incubated overnight at 4°C with primary' antibodies (see Table 3) and HRP -conjugated secondary' antibodies for 2 hours, and proteins were visualized using ChemiDoc Imaging System (BioRad).

[0110] Immunohistochemistry / Immunofluorescence

[0111] Briefly, tissues of PDX tumors (treated with vehicle control or inhibitors [L- NMMA, alpelisib, L-NMMA+alpelisib]) as well as baseline and end-of-therapy tissue from nine available phase 2 dose patients (three responders and six nonresponders) were evaluated by IHC for iNOS, phospho-Akt (Ser473), and PTEN expression. FFPE tissues were prepared and underwent xylene deparaffmization, ethanol rehydration, antigen retrieval, fixation with hydrogen peroxide, blocking, antibody incubation, chromogen reaction, and counterstained with hematoxylin, slides were mounted and imaged under bright field microscopy. iNOS staining was analyzed by H-score analysis using ImageJ plugin IHC Profiler. For immunofluorescence analysis, same FFPE tissues underwent deparaffmization and antigen retrieval as previously described with no hydrogen peroxide fixation, incubation with primary / secondary antibodies. Slides were mounted with Antifade Mounting Medium with DAPI (Vector Laboratories) and sealed with coverslips.

[0112] In vivo Experiments

[0113] All animal procedures were approved by7the Houston Methodist Hospital Research Institute Animal Care and Review Office. The maximal tumor volume permitted in our study is 2000 mm3 as calculated from caliper tumor measurements. All procedures were performed in accordance with the protocol and none of the tumor volumes in our study exceeded 2000 mm3 at the time point before the last time point when the studies were terminated. Some tumors exceeded this limit at the last time point and these animals wereDocket No. 38358.0004P1 immediately euthanized. In vivo experiments were conducted in four human MpBC PDX models (BCM-4664, BCM-3807, PIM-010, and PIM-084). PDXs were implanted into cleared mammary fat pad of SCID / Beige mice (Envigo). When tumors reached an average volume between 150 and 250 mm3, mice were randomized into treatment arms. Mouse weights were record and tumor volume was measured and calculated [0.5 x (long dimension) x (short dimension)2] twice weekly. Tumor volume change was calculated by dividing change in tumor volume at last measurement by initial tumor volume. Regiment treatment design followed either two or three, 2-week cycles of docetaxel (20 mg / kg intraperitoneal on day 1), NOS inhibition therapy (L-NMMA [400 mg / kg oral gavage on day 2 and 9, 200 mg / kg oral gavage on days 3-6 and 10-13] + amlodipine [10 mg / kg intraperitoneal injection on days 2-6 and 9-13]), PI3K inhibitor alpelisib (35 mg / kg oral gavage on days 2-6 and 9-13), or the combination of therapies as indicated. Caliper measurements were taken twice a week.

[0114] Breast Cancer Stem Cell Assays:

[0115] Mice implanted with BCM-3807 PDX tumors of 150-250 mm3 were randomized to receive one cycle (two weeks) of docetaxel, docetaxel+L-NMMA or alpelisib, and triple combination. Dissociated tumors were transplanted with Matrigel (1 :1) into the fatpad of SCID / Beige mice. For limiting dilution assay experiment in Figure 12, we transplanted 30,000 and 50,0000 PDX-3807 tumor cells into the fat-pad of SCID / Beige mice (n=5-10 per group) from tumors treated for two weeks with docetaxel, docetaxel+L-NMMA or alpelisib, and triple combination. For limiting dilution assay experiment in FIG. 11, BCM- 3807 tumors from docetaxel and triple combination (docetaxel+L-NMMA+alpelisib) treated mice (14 days) were dissociated and pooled. A total of 1000, 10000, and 100000 cells from each group were transplanted into the mammary gland fat-pad of 4- to 6-week-old mice (n=13-15 mice / group). Tumor incidence for both experiments was reported at 12 weeks posttransplantation. Stem cell frequency fractions and overall statistical analysis comparing differences in stem cell frequencies between treatment groups was evaluated using available ELDA software ( bioinf.wehi.edu.au / software / elda / ).

[0116] For flow cytometry, dissociated tumor cells post-treatment (two weeks) were prepared at a concentration of 1 million cells per 0. 1 ml in Hank's Balanced Salt Solution (HBSS) supplemented with 2% fetal bovine serum (HBSS+). These cells underwent labeling procedures using fluorophore-conjugated antibodies, including CD44-APC (at a 1 : 10 dilution), CD24-PECY7 (at a 1:40 dilution) or CD24-FITC (at a 1 : 10 dilution), and H2kD-PE (at a 1:40 dilution) to eliminate mouse cells. Labeling was conducted either on ice for 15Docket No. 38358.0004P1 minutes or at 37°C for 45 minutes, following the manufacturer's protocol for the Aldefluor kit (StemCell Technologies, B.C., Canada). Additionally, post-Aldefluor or DEAB labeling, H2kD-PE staining was performed on ice for 15 minutes in 0.1 mL HBSS+. Subsequently, cells were washed and treated with propidium iodide at a concentration of 10 pg / mL prior to analysis or sorting using a four-laser FACS Anall system (BD Biosciences, San Jose, C.A., USA). Debris and doublets were excluded based on side scatter and forward scatter parameters, while propidium iodide staining was utilized to eliminate non-viable cells. Tumor cells negative for H2kD were further characterized for CD44 and CD24 expression or aldehyde dehydrogenase activity. Data analysis was conducted using FACS Diva software (BD Biosciences) and FCS Express (Denovo Software).

[0117] For MSFE assays, dissociated tumor cells (60,000) were plated in MammoCult Media (Stem Cells) in low-adherent 6-well and 24-well plates. Mammospheres were imaged and counted using GelCount imaging and software system (Oxford Optronix Ltd.). For secondary MSFE, mammospheres were collected, dissociated with trypsin, filtered, counted, and replated at 60,000 cells / mL MammoCult Media.

[0118] Statistical Analysis

[0119] Two-tailed Student t test or Mann- Whitney test, with the mean values and the SD calculated for each group was performed for comparisons between two groups. One-way ANOVA was performed for multiple group comparisons. Two-way ANOVA was used for all animal experiments. To account for multiple comparisons, Tukey's multiple comparison tests for one-way ANOVA and Bonferroni post tests for two-way ANOVA were performed. Analysis was conducted using Graphpad Prism 5.0 (Graphpad Software Inc.) and Stata V16.1 (StataCorp). In all cases, a two-tailed P value < 0.05 was considered statistically significant.

[0120] Results

[0121] Evidence of clinical activity of NOS inhibition with L-NMMA combined with taxane in MpBC patients

[0122] In a phase I / II clinical trial of L-NMMA plus taxane for treating patients with chemorefractory, locally advanced breast cancer (LABC) or metastatic TNBC13, 35 TNBC patients were recruited, and 15 patients had MpBC (Phase IB, n= 4; Phase 2, n=l l); 86.6% (13 / 15) patients had metastatic breast cancer (MBC), with a median of 2 prior lines of therapy (range 0-5) and 13.3% (2 / 15) had anthracycline-refractory locally advanced breast cancer (LABC). (FIG. 1A, FIG. 2A). The median age of the cohort was 62 years (35-75 years). Two patients were excluded from analysis due to adverse events unrelated to treatment. TheDocket No. 38358.0004P1 clinical benefit rate (CBR) was 46% (6 / 13); the overall response rate (ORR) was 23% (3 / 13) with one partial response (PR) in metastatic TNBC, one pathological complete response (pCR) and one PR in LABC (FIG. 1A, FIG. 2A). Grade 3 or more toxicity was seen in 15% (2 / 13) patients; however, none were attributed to L-NMMA and were likely related to the standard of care docetaxel. The median progression free survival and median overall survival for MBC patients were 4.5 months (range 3-7 months) and 12.8 months, respectively. The clinical characteristics of all patients are listed in Tables 5 and 6.Table 5. Overview of MpBC Patient Characteristics analyzed from Phase 1B / 2 Clinical TrialDocket No. 38358.0004P1Table 6. Association among Ethnicity, Body Mass Index (BMI), and Pathological Features with Response.

[0123] The patient with pCR at the time of trial enrollment had a bulky primary mass with extensive skin ulceration which was refractory to anthracy cline and achieved a complete response after LNMMA therapy (FIG. 1B-D) and showed the highest intensity of iNOS expression at baseline (BL) (FIG. 1A, D, E). For all responders, iNOS expression had a significant reduction at end-of-treatment (EOT), underscoring the importance of NOS inhibition in combined therapy for improving clinical response. In three out of six nonresponders, iNOS expression increased from BL to EOT with statistical significance in two non-responders (p=0.002, p<0.001) (FIG. IE). Since MpBC tumors typically have genomic alterations in PI3K signaling along with increased iNOS activation, available next generation sequencing data of TP53, PIK3CA, PIK.3R1, and AKT1 mutations for 7 / 13 patients was evaluated (FIG. 2B). 4 / 7 patients had TP53 mutations, one with progressive disease (PD), two with stable disease (SD), and one with a partial response. 2 / 7 patients had PIK3CA mutations and were nonresponders with disease progression. Two patients with SD had AKT1 mutations, and one with a concomitant PIK3R1 mutation. Next generation sequencing for 6 / 13 patients was not available because either the analysis was not conducted or because the data was not available to us for further analysis.

[0124] These clinical findings suggested that targeting iNOS may be a potential therapeutic strategy’ to augment the efficacy of taxane and potentially targeted therapies inDocket No. 38358.0004P1 chemoresistant MpBC. To fully define the relevance of targeting iNOS in MpBC, we analyzed available transcriptomic data from The Cancer Genome Atlas (TCGA) cBioPortal Combined Dataset (n= 179290) (cbioportal.org), which showed that ‘MpBC’ was the top breast cancer subtype and 4th highest cancer to harbor NOS2 genomic aberrations (FIG. IF). TCGA is limited in the number of MpBC cases and associated clinicopathological characteristics, thereby making it difficult to evaluate any potential clinical correlations with NOS expression. To further understand the clinical importance of NOS in MpBC, datasets from the ARTEMIS clinical trial that recruited patients with newly diagnosed TNBC and MpBC were analyzed and provided an avenue to investigate the genomic landscape of patients with these cancers. Transcriptomic RNA-seq analysis of treatment-naive TNBC tumors enriched with MpBC histology' and metaplastic-like characteristics from ARTEMIS found that MpBC+metaplastic-like tumors had higher tumor NOS2 expression than non- MpTNBC (FIG. 1G), further supporting our findings from the TCGA cBioPortal Combined Dataset (FIG. IF). More significantly, high tumor N0S2 mRNA expression was correlated with significantly worse metastasis-free survival (MFS) [p=0.042], indicating the importance of inhibiting iNOS to improve clinical outcome in patients w ith MpBC (Fig. 1H).

[0125] The transcriptomes of MpBC were compared to invasive ductal carcinoma (IDC) tumors from TCGA (FIG. II, FIG. 2C). Relative to IDC, the top enriched pathways in MpBC tumors include epithelial -to-mesenchymal transition (EMT), hypoxia, PI3K-Akt signaling pathway, and extracellular matrix regulation (FIG. II). These findings further support increased signaling of iNOS in MpBC considering the established link between HIF- la-induced hypoxia and enhanced iNOS function, the proposed alteration of EMT programming by iNOS activation, and the reported stimulation of PI3K pathway by iNOS- derived NO regardless of PIK3CA mutation status.

[0126] Enhanced co-activation of iNOS and PI3K signaling in MpBC tumors

[0127] To understand the biological relationship between iNOS and PI3K signaling pathways, the STRING database (string-db.org) was queried for the term ’NOS2’ for high confidence (score>0.7) protein-protein interactions. Results showed that NOS2 functionally interacts with AKT1 with a highly enrichment p-value (0.000538) (FIG. 3A). Biological interactions of NOS with EMT, PI3K, and hypoxia-related genes (p=1.21e-08) (FIG. 4A) strongly support the clinical relevance of NOS and PI3K pathways and their potential interaction in MpBC. Mutual exclusivity' analysis in data from cBioPortal Combined Studies (n=l 79290) and PanCancer Atlas Studies (n=76639) of TCGA showed that genomicDocket No. 38358.0004P1 alterations in NOS2 significantly co-occur with those of PIK3CA, PTEN, and AKT1 genes (all p<0.001) in multiple cancer types (FIG. 3B, FIG. 4B). Similar significant co-occurrence of NOS2 and AKT1 gene alterations were found in the Breast Cancer cohort from TCGA (n=8644) (p<0.001) suggesting that iNOS and PI3K signaling are complementary and have collaborative oncological function (FIG. 4C). The significance of these NOS2 genetic alterations in relation to disease pathogenesis is currently unknown; however, the significant co-occurrence between NOS2 and PI3K signaling genes implies that these pathways may be influencing tumorigenesis by utilizing similar mechanisms in multiple cancer types, including MpBC.

[0128] Next, the distribution of iNOS and active Akt co-expression was assessed among breast cancer subtypes: ER+, HER2+, TNBC, and MpBC. The expression of iNOS and phosphorylated forms of Akt (Ser473 and Thr308) was assessed in 15 breast cancer cell lines and human mammary epithelial cell line MCF10A. Relative to non-metaplastic TNBC cell lines, MpBC cell lines had exceptionally elevated protein levels of iNOS (p=0.0426) and phospho-Akt (Thr308) (p=0.0223) (FIG. 3C, 3D). Immunohistochemistry was used to evaluate the protein expression of iNOS and phospho-Akt in tissues from breast cancer PDX models. MpBC PDXs predominantly had more co-expression of high / moderate staining of iNOS and phospho-Akt relative to other breast cancer PDX subtypes (FIG. 3E).

[0129] Droplet digital polymerase chain reaction (ddPCR) was utilized to evaluate whether these corresponding breast cancer PDXs harbored the RPL39 A14V mutation, associated with iNOS activation, and PIK3CA hotspot mutations (E542K, E545K, H1047R, H1047L). The RPL39 A14V mutation was detected in four out of six (66%) MpBC PDX models versus one out of 21 (4.7%) TNBC PDX models (p=0.004) and no mutations were found in ER+ or HER2+ models. PIK3C A hotspot mutations w ere found in 3 / 6 (50%) MpBC PDX models versus 4 / 21 (19%) TNBC, 2 / 5 ER+, and 2 / 3 HER2+ PDX models (FIG. 3E, 3F). Specifically, the MpBC PDX models PIM-010 and PIM-084 were RPL39 / PIK3CA- mutated and had corresponding expression of iNOS (high) and phospho-Akt (Ser473). Consistent with the protein levels, NOS2 mRNA expression was substantially higher in MpBC tumors compared to other breast cancer subtypes by performing quantitative PCR (qPCR) analysis in the corresponding 35 PDX models (FIG. 3G). Overall, these findings underscore the significance of iNOS as a molecular target and propose its inhibition alone or together with the co-activated PI3K as a potential therapeutic strategy for MpBC.

[0130] NOS inhibition acts synergistically with PI3K inhibitor in MpBC cell linesDocket No. 38358.0004P1

[0131] To explore whether combined inhibition of iNOS by L-NMMA and PI3K signaling by alpelisib is synergistic in MpBC models, a small cell line screen was performed with MpBC cell lines BT549, Hs578T, SUM159 and HCC1806. These cell lines were derived from primary carcinosarcomas and / or exhibited metaplastic features by pathology (FIG. 5A)

[0132] The IC50 values of alpelisib in each MpBC cell line are summarized (FIG. 5B). Notably, three MpBC cell lines (SUM159, Hs578T, and BT549) were sensitive to alpelisib and achieved IC50 concentrations under the threshold for a physiological achievable dose (5.6 pM). based on a previous phase lb trial23. Cell viability assays revealed that combining L-NMMA and alpelisib was synergistic in SUM159 and Hs578T cell lines, which harbor PIK3CA and PIK3R1 mutations, respectively (combination index [CI] <1). L-NMMA and alpelisib combination therapy was antagonistic in BT549 (PTEN mutated) and HCC1806 (PIK3CA / PTEN wild-type) cell lines (CI>1) (FIG. 6A). The synergistic effect seen in SUM159 and Hs578T cell lines was also confirmed with anchorage-dependent colony formation (FIG. 5C) and caspase 3 / 7 activation assays (FIG. 5D). To confirm this observation, clones of SUM159 cells with CRISPR / CAS9-mediated knockout of NOS2 (NOS2KO) were generated, confirmed by the absence of iNOS protein in immunoblots and reduced production of nitrite / nitrate in NOS2KO clones relative to parental SUM159 cells (FIG. 6B, FIG. 5E). The absence of iNOS in the NOS2KO clones was associated with substantially reduced expression of phospho-Akt (Ser473 / Thr308), suggesting that NOS downregulation could impair Akt activation. Further, consistent with the pharmacological inhibition, ablation of NOS in SUM159 NOS2KO clones led to an enhanced sensitivity to alpelisib (FIG. 6A-C). Finally, pharmacological inhibition of NOS resulted in decreased phosphorylation of Akt and S6 in MpBC cell lines SUM159 and Hs578T, further reduced by the addition of PI3K inhibition, resembling the synergistic effect of two inhibitors on the survival of the same cell lines (Fig. 6A, 6D, FIG. 5F). The combination therapy did not significantly reduce phosphorylation of Akt and S6 in BT549 cell line (FIG. 6D, FIG. 5F). The correlation between differential response to NOS inhibition and variability in S- nitrosoglutathione reductase (GSNOR) expression was evaluated. Cell lines that were responsive to combination therapy (SUM159 and Hs578T) had a relatively reduced expression of GSNOR, compared to cell lines that were not responsive to combination therapy (HCC1806 and BT549) (FIG. 6E).Docket No. 38358.0004P1

[0133] To better understand the underlying mechanism of the synergistic cytotoxic effect of NOS and PI3K inactivation on MpBC cells, cellular events affected by single-agent treatment were investigated. PI3K inhibitors, such as alpelisib, induce DNA-damage via nucleotide depletion and are synergistic with PARP inhibitors in BRCA-mutant breast cancers. Data showed that NOS inhibition alone induced DNA damage and the combination of LNMMA with alpelisib augmented this effect in PI3KCA / PIK3R1 mutated cells (SUM159 / Hs578T), as shown by increased comet tail moment (FIG. 6F), enhanced yH2AX signal with decreased Rad51 foci formation in immunofluorescence analysis, and timedependent increase in yH2AX protein levels with concomitant decrease in Rad51 expression (FIG. 5G-H). In contrast, in PTEN-deleted BT549 cells, NOS inhibition was unable to enhance the DNA damaging effects of alpelisib (FIG. 6F, FIG. 51). The involvement of DNA damage in the synergistic cytotoxic effect in the presence of PIK3CA mutations was supported by enhanced nucleotide depletion observed in the presence of the combination therapy (L-NMMA+alpehsib) in SUMI 59 cells but not in BT549 cells using an HIV reverse transcriptase (RT)-based dNTP assay (FIG. 6J-K). These results suggest that L-NMMA augments the effect of PI3K inhibitor in inducing DNA damage in MpBC cells with PIK3CA or PIK3R1 mutations by enhanced nucleotide depletion, leading to increased apoptosis and enhanced sensitivity of MpBC cells to dual NOS and PI3K inhibition.

[0134] NOS inhibition sensitizes MpBC tumors to PI3K inhibitor and taxane chemotherapy in vivo

[0135] To support in vitro studies, the effect of combined NOS and PI3K inhibitor treatment in MpBC PDX tumors was evaluated. In orthotopic MpBC PDX models, mice were randomly assigned to one of four groups to receive vehicle, L-NMMA, alpelisib, or combination of L-NMMA and alpelisib (FIG. 7A). The combination of L-NMMA+alpelisib was well tolerated as indicated by mouse body weight over time (FIG. 7A-B). L- NMMA+alpelisib therapy resulted in a significant inhibition of tumor growth relative to single-agent alpelisib therapy in PIK3CA-mutant MpBC PDX models (BCM-3807 [p=0.0456], PIM-010 [p=0.0079], and PIM-084 [p=0.016]), (FIG. 7B-D). In BCM-4664 (PIK3CA-wild-type) PDX model, the addition of L-NMMA to alpelisib did not significantly reduce tumor volume relative to single-agent alpelisib therapy (p=0.1275) (FIG. 7E). However, a trend toward enhanced tumor response to combination therapy in this PDX was observed, suggesting that NOS inhibition may augment the efficacy of PI3K inhibition regardless of PIK3CA mutation status. Combination therapy also resulted in a significantDocket No. 38358.0004P1 decrease in protein levels of iNOS and phospho- Akt, as shown by quantification of Immunohistochemistry (IHC) H-score analysis (FIG. 8C-E) and the enhanced expression of cleaved caspase 3 in BCM-3807 tissues (FIG. 8F).

[0136] Driven by the benefit of the combination therapy (L-NMMA plus taxane) in the clinical trial with the chemorefractory MpBC patients (FIG. 1) and considering that MpBCs have a worse pCR rate than non-MpTNBC to standard-of-care chemotherapeutic regimens 26, we next evaluated whether L-NMMA alone and its combination with alpelisib was effective at augmenting the efficacy of chemotherapy in chemoresistant MpBC tumors (FIG. 8G). PDX models BCM-4664 (PIK3CA wild-type) and BCM-3807 (PIK3CA E545K) were treated with three cycles of taxane + / - targeted therapies. The triple combination therapy (L-NMMA+alpelisib+docetaxel) reduced the volume of BCM-4664 tumors and significantly improved OS of recipient mice in comparison to docetaxel+alpelisib therapy (p=0.0432) and docetaxel+L-NMMA therapy (p=0.0007) (FIG. 7F, H). In BCM-3807 model, the combination therapy eradicated the tumors in 5 out of 7 animals with no detectable tumor regrowth after treatment discontinuation (Day 43) (FIG. 7G) and extended OS in comparison to docetaxel-only treatment arm (p=0.0192) (FIG. 71). Cell viability assays also confirm our findings showing that NOS2KO SUM159 clones were more sensitive to docetaxel treatment than control SUM 159 cells. (FIG. 7H).

[0137] NOS inhibition reverses EMT transition in MpBC cell lines and PDX models

[0138] To define the molecular mechanism of the synergistic effect of NOS and PI3K inhibition in MpBC, RNA-Seq / GSEA was performed in MpBC PDX tumors following treatment with dual combination therapy. GSEA indicated that L-NMMA+alpelisib treated tumors were enriched with pathways associated with cellular epithelization and differentiation, including formation of cornified envelopment, keratinization, and negatively enriched for hypoxia and EMT pathways (FIG. 9A, FIG. 10A). This analysis suggested that combined NOS and PI3K inhibition may have enabled MpBC tumor cells to undergo reversal of EMT and enhanced cellular epithelization. Immunofluorescence analysis of BCM-3807 and PIM-010 treated tumors was used to determine expression of the epithelial marker E- cadherin and ZEB 127. L-NMMA single-agent and L-NMMA+alpelisib treated tumors had increased expression of E-cadherin with an associated decrease in ZEB1 (FIG. 9B-C, FIG. 10B-C), suggesting that NOS inhibition may have induced a reversal of EMT, as suggested in our previous study using TNBC modelsl 1. In MpBC cell lines (Hs578T and SUM159), inhibition of NOS alone by L-NMMA and combined with the PI3K inhibitor alpelisib led toDocket No. 38358.0004P1 reduced protein levels of ZEB 1 and dow nstream CHK1, with an increased expression of E- cadherin and zonula occludens-1 (ZO-1) (FIG. 9D, FIG. 10D). Consistent w ith the repressive effect of NOS inhibitor on EMT, SUMI 59 cells with NOS2 knockout were more compact with a cobblestone-like morphology compared with parental cells that had decreased adhesions, spindle-like structures, and enlarged protrusions (FIG. 9E). This result was further supported by decreased protein levels of iNOS, phospho- Akt, HIF-la, Zebl, vimentin, and latent TGFp, increased expression of E-cadherin and ZO-1 and decreased migratory capacity of N0S2K0 clones relative to parental SUM159 cells (FIG. 9F, FIG. 10E-F).

[0139] To evaluate how NOS inhibition caused reversal of EMT in MpBC cells, RNA-sequencing was performed in SUM159 NOS2KO cells. Gene ontology analysis of the transcriptomic data revealed a list of significantly downregulated genes in NOS2KO cells associated with EMT induction and CSC maintenance including LCN2, TGFB1, GLI1, NOTCH1, COL17A1 (FIG. 9G). In addition, the NOS2KO clones had corresponding enrichment of pathways such as homophilic cell adhesion, epithelial differentiation, and extracellular matrix organization (FIG. 9H). The expression of two of the downregulated genes and potent drivers of EMT (TGFB1 and LCN2) w as assessed by qPCR and immunoblotting and showed substantial decrease in the mRNA and protein levels in SUM159 NOS2KO clones, indicating the involvement of iNOS in the transcriptional regulation of these genes (FIG. 9I-K). The central role of TGFP in mediating the effect of NOS on EMT in MpBC by treating SUMI 59 NOS2KO cells with TGFP was investigated further. As expected, activation of TGFp completely reversed the epithelial transformation induced by the downregulation of NOS in SUM159 cells, as indicated by the prevalence of mesenchymal morphology and strong upregulation of the mesenchymal markers Vimentin and ZEB1 in TGFP-treated NOS2KO cells (FIG. 10G-H). These findings led to the investigation of transcriptional machinery regulating the expression of TGFB1 and LCN2 to identify direct targets of NOS in MpBC cells, including factors that act on AP-1 response elements like c- Jun and Fos.

[0140] According to our STRING database query, NOS2 had a significant biological interaction with JUN (FIG. 3A), suggesting that NO may trigger the activation of AP-1 transcription factors, leading to altered expression of EMT effectors and overall altered EMT programming. To test this, the factors necessary for the transcription of LCN2 / TGFB1 in MpBC cells were investigated by treating SUM159 cells with siRNAs specific to CREB3, XBP1, FOS, and JUN (FIG. 101).Docket No. 38358.0004P1

[0141] JUN knockdown resulted in the strongest associated decrease in the mRNA levels of TGFB1 and LCN2, suggesting that c-Jun may be the direct mediator of the transcriptional effects of iNOS on EMT by regulating TGFB1 and LCN2 (FIG. 9L-M, FIG. 101). The direct effect of iNOS on c-Jun transcriptional complex in MpBC is further supported by previous studies showing that the regulator of c-Jun, c-Jun N-terminal kinase (JNK) is subject to S-nitrosylation-mediated activation in cardiac dysfunction and fibrosis. These studies suggest that in MpBC cells, NO may increase the transcriptional activity of JUN by enhancing the function of JNK through S-nitrosylation. In relation to this proposed mechanistic model, NOS2KO cells had a corresponding decrease in JNK-specific phosphorylation of c-Jun at Ser63 / Ser73 sites with no associated change in expression of total c-Jun (FIG. 9N) and a decrease in S-nitrosylation of JNK (SNO-JNK) (FIG. 90). These findings were also confirmed in BT549 cells that underwent CRISPR Cas9-mediated targeting of NOS2 (FIG. 10J-L). EMT reversal was characterized by reduced activation of c- JUN via phosphorylation, and reduced protein expression of EMT effectors TGF(3 and LCN2 in Hs578T cells treated with siRNA targeting NOS2 (FIG. 10M-O).

[0142] L-NMMA and alpelisib combined with taxane chemotherapy effectively targets breast CSCs

[0143] EMT programming can induce the acquisition of breast cancer stem cell (BCSC) properties and MpBC tumors are highly enriched with chemoresistant BCSC populations. RNA-seq analysis showed downregulation of genes involved in CSC maintenance in SUM159 NOS2KO cells and increased chemosensitivity of MpBC tumors when treated with NOS and PI3K inhibitors (FIG. 7 and FIG. 9). Based on this finding, the efficacy of a combination of NOS / PI3K inactivation with taxane chemotherapy targeting BCSCs was evaluated.

[0144] For this, mice bearing BCM-3807 PDX tumors were stratified among eight treatment groups: vehicle control, single-agent therapy (L-NMMA, alpelisib. docetaxel), dual-agent therapy (docetaxel+L-NMMA. docetaxel+alpelisib). and tnple-combination (docetaxel+L-NMMA+alpelisib). Flow-cytometric analysis for BCSC markers, mammosphere-formation efficiency (MSFE) assays, and limiting dilution assays were conducted following a 14-day treatment period (FIG. 11A).

[0145] Primary and secondary MSFE was significantly decreased in tumors with triple combination therapy compared with vehicle treatment (Fig. 6B, C). Triple combination therapy significantly reduced the formation of secondary7mammospheres compared to doubleDocket No. 38358.0004P1 combination therapy (FIG. 11C). Triple combination treatment also reduced the CD44+ / CD24- subpopulation of cells (BCSC marker) and reduced tumor cells capable of tumor initiation, as shown by limiting dilution assay. (FIG. 11D-E, FIG. 12A). To further evaluate whether triple combination therapy was effective at targeting cells with tumorinitiating potential that may be resident post-docetaxel therapy, limiting dilutions cells from BCM-3807 tumors treated for 14 days with either docetaxel or triple combination therapy were transplanted into mammary' fat pads of SCID / Beige mice. After 12 weeks, secondary' transplantation of cells derived from triple combination therapy resulted in a 17-fold loss of tumor-initiating ability compared to docetaxel therapy only (FIG. 11F).

[0146] Flow cy tometric analysis of BCSC marker aldehyde dehydrogenase (ALDH1) was performed in cells from responsive BCM-3807 tumors following single-agent and dualagent treatment. ALDH1+ cells were significantly decreased in tumors after L- NMMA+alpelisib compared to vehicle control (p=0.0006), L-NMMA (p=0.036), and alpelisib (p=0.047) (FIG. 11G-H). Comparable results by immunofluorescence analysis of ALDH1 in responsive PDX BCM-3807 and PIM-010 tumors following single-agent and dual agent treatment w as observed. ALDH1 expression w as significantly decreased in dual agent treated BCM-3807 (pO.OOOl) and PIM-010 (p<0.0001) tumors relative to vehicle control treated tumors (FIG. 12B-E). Overall, the data suggests that combined NOS and PI3K inhibition is capable of targeting chemoresistant and tumor-initiating population of breast cancer cells within MpBC tumors, leading to their increased chemosensitivity'.

[0147] NOS inhibition reverses EMT in MpBC human tumor biopsies

[0148] To confirm in vitro and in vivo findings revealing that NOS inhibition reverses EMT and enhances tumor differentiation, immunofluorescence analysis was used to evaluate the expression of epithelial marker E-cadherin, ZEB1, and ALDH1 in responder and nonresponder tumor biopsies at BL and EOT from the L-NMMA+taxane clinical trial discussed in FIG. 1. Corresponding hematoxylin and eosin staining was completed using BL and EOT biopsies representative of non-responder and responder tissues (FIG. 13A-B) in FIG. 14. Zebl showed a statistically significant decrease in all responders (p<0.05) with no significant change in non-responders (FIG. 13A-C). Similarly, increase in E-cadherin from BL to EOT was statistically significant in all responders (p<0.05), whereas it had no significant change in the non-responders (FIG. 13A, B, D). ALDH1, a marker of mammary stem cell and tumor initiation, showed a statistically significant decrease in tw o out of the three responders, from BL to EOT (p<0.05), but no significant change in the non-responders. All non-respondersDocket No. 38358.0004P1 had an elevated baseline level of ALDH1 as compared to responders (FIG. 13A, B, E). Collectively, these findings suggest that iNOS elicits its oncogenic activity in MpBC by acting on a potent activator of EMT and tumor dedifferentiation (FIG. 13F), providing an additional line of evidence of NOS as a highly valuable molecular target in clinical management of MpBC. This study showed that a quasi-mesenchymal / mesenchymal state, iNOS from MpBC cells can produce NO, leading to the attachment of a S-nitrosyl group to a reactive thiol group in JNK, forming a S-nitrosothiol (SNO). SNO-JNK activates the kinase, leading to phosphorylation of AP-1 proteins, such as c-Jun transcription factor, resulting in enhanced expression of EMT mediators TGFB1 and LCN2. TGF[3 can activate PI3K / Akt signaling by stimulating Type I and II serine / threonine kinase receptor complex (TpRI / TpRII), causing TpRI to associate with p85, mediating Akt activation.35 NO can also activate pro-survival signaling pathways, such as PI3K, that influence activation of JNK in a mechanism independent of S-nitrosylation. Under NOS inhibition, reduced activation of PI3K signaling and decreased formation of SNO-JNK result in less activation of c-Jun, and decreased mRNA expression of TGFB1 and LCN2. The reduced expression of EMT mediators results in MpBC cells to differentiate back to an epithelial-like state, leading to increased susceptibility to conventional taxane chemotherapy (FIG. 13G).

[0149] Discussion

[0150] Despite the use of conventional treatment with surgery and chemo / radiotherapies to treat MpBC, clinical outcomes remain poor. The aggressiveness of MpBC has been attributed to enrichment of EMT / CSC features, triggered by the activation of oncogenic pathways, including iNOS and PI3K signaling. The findings presented herein demonstrated that human MpBC tumors show' enhanced co-activation of iNOS and PI3K signaling, and a strong association betw een increased expression of NOS2 and a w orse MFS in patients with TNBC and MpBC. This initial analysis of human tumors enabled revealed the potential for dual targeting of the hyperactivated NOS and PI3K pathways as a rational therapeutic strategy to treat chemoresistant MpBC. NOS inhibition augmented the efficacy of PI3K inhibitor alpelisib in MpBC in vitro and in vivo models. Combined NOS and PI3K inhibition further sensitized MpBC tumors to chemotherapy and improved OS regardless of PIK3CA mutation status. In MpBC cell line studies, combined PI3K and NOS inhibition had an antagonist effect in BT549 (PTEN mutated) and HCC1806 (PIK3CA / PTEN wild type) cell lines. BT549 likely had a poor response to combined NOS and alpha-isoform specific PI3K inhibition due to its lack of PTEN. This deficiency may have led these cells to be moreDocket No. 38358.0004P1 dependent on the pl 10(3 PI3K isoform. Furthermore, the absence of PTEN suggests that BT549 cells may rely less on NO for oncogenic signaling, as PTEN's post-translational inactivation via S-nitrosylation would not be possible. HCC1806 had a poor response to combination therapy likely due to its low activation of iNOS and PI3K signaling, as exhibited in FIG. 3C as well as its relatively higher expression of GSNOR seen in FIG. 6F.

[0151] NOS inhibition overcomes resistance and augments the efficacy of alpelisib and taxane therapy by primarily inhibiting EMT and decreasing sternness, leading to enhanced tumor cell differentiation. The suppression of EMT in MpBC appears to contribute to the synergistic cytotoxicity and DNA damage elicited by dual NOS and PI3K inhibition in MpBC tumors. It may explain the delayed growth of MpBC tumors in mice treated with L- NMMA and taxane-based chemotherapy, the benefit of MpBC patients who received the same type of therapy, and the significant association of low NOS expression with longer MFS that we observed in the cohort with MpBC patients (FIG. 1H).

[0152] In MpBC cells, NOS inhibition decreases production of NO, thus reducing formation of SNO-JNK. Reduced SNO-JNK substantially impaired phosphorylation of AP-1 transcription factor c-Jun and decreased the expression of EMT mediators, TGF(3 and lipocalin. resulting in tumor cell epithelization. Furthermore, combining L-NMMA and alpelisib with taxane was superior to taxane alone at targeting chemoresistant CSC populations, resulting in significantly decreased overall tumor burden and improved OS. Additionally, combined targeting of NOS and PI3K reduced cell proliferation, thereby providing a two-pronged attack by reducing invasion through inhibiting EMT and decreasing proliferation of MpBC tumor cells that may colonize metastatic sites. Lastly, in a phase lb / 2 clinical trial assessing the efficacy of L-NMMA combined with taxane to treating patients with chemorefractory, LABC or metastatic TNBC, tumor biopsies of responder / non- responder MpBC tumors w ere evaluated. In responder tumor biopsies at EOT, there was an associated enhancement in tissue differentiation / epithelization, along with a decreased expression of CSC marker ALDH1 and iNOS. The sample size of MpBC human tumor biopsies in the study was small (n=13), however MpBC is a rare and highly aggressive pathology'. Although the investigation is confined by the quantity of MpBC PDXs, PDXs exhibiting favorable responses to combination therapy primarily exhibited heightened protein expression of iNOS and pAkt, in conjunction with presence of RPL39 / PIK3CA mutations. Additionally, they were characterized as matrix-producing metaplastic (PIM-010 and PIM- 084) and squamous metaplastic (BCM-3807) carcinomas. Using in vitro / in vivo approachesDocket No. 38358.0004P1 and validating with human MpBC tissue, data showed that inhibition of tumor-intrinsic NOS can overcome chemoresistance via induced tumor differentiation.

[0153] In other disease models, it has been reported that iNOS-derived NO can enhance the expression of genes associated with fibrosis through S-nitrosylation-mediated activation of JNK. An activated form of SNO-JNK phosphorylates c-Jun. resulting in transcriptional activation of AP-1 downstream genes. The data provided herein delineated the relationship between iNOS and the major mediators of EMT, thus proposing a mechanism of how tumor differentiation processes can be modulated for therapeutic benefit, in cancers like MpBC. Initial findings using the TCGA database suggested that there may be enhanced iNOS activation in other cancers, such as endometrial and ovarian cancers. Increased co-occurrence of mutations in genes from iNOS and PI3K signaling in multiple cancer types and the findings presented herein suggest that NOS is upstream of PI3K signaling proteins. In highgrade ovarian cancers, nearly 70% of all cases have hyperactivation of PI3K signaling pathway, high iNOS expression is predominant in these tumors and may be associated with resistance to platinum-based chemotherapies and enhanced metastatic capacity mediated by EMT. Co-targeting NOS and PI3K may be a rational therapeutic strategy' to treat MpBC.

[0154] The influence of iNOS and nitric oxide on activation of PI3K signaling regardless of PIK3CA mutation status is documented. Additionally, iNOS has been shown to modulate PI3K / Akt signaling through various S-nitrosylation mechanisms. For example, in melanoma models, iNOS -generated NO can reversibly S-nitrosylate the TSC2 protein, disrupting TSC2 / TSC1 dimerization, leading to mTOR activation and increased melanoma cell proliferation. INOS-derived NO can also S-nitrosylate PTEN protein, reducing PTEN phosphatase activity and promoting PI3K / Akt signaling. Finally, in breast cancer cells, iNOS- associated Akt activation necessitates TIMP1 protein nitration and TIMP1 and CD63 proteinprotein interactions.

[0155] Tipping the EMT programming scale towards more of an epithelial / proliferative state and targeting CSCs may enhance tumor sensitivity to targeted therapies and chemo / radiotherapies. The tumor microenvironment (TME) is a critical source for factors like hypoxia (HIFla, NO), and signaling proteins derived from cells such as cancer-associated fibroblasts and macrophages (TGFty LCN2, IL-6, IL-1J3, etc.) that influence EMT.

[0156] The data presented herein evaluated how- targeting tumor-intrinsic iNOS could influence MpBC differentiation and therapy response. Sequencing analysis of MpBC cellsDocket No. 38358.0004P1 lacking N0S2 revealed a significant decrease in the expression of pro-inflammatory genes (CXCL8, ILIA, IL1B, IL1RL1), suggesting that there may be crosstalk between tumor- intrinsic iNOS and TME-derived inflammatory cells that may influence tumor EMT programming. Since MpBC are typically TNBC, analysis of available syngeneic models of this breast cancer subtype may assist to better characterize aspects of NOS function involving the TME and impacting the clinical effects of NOS targeted therapies. Example 2: Phase II of Alpelisib with iNOS Inhibitor and Nab-paclitaxel in Patients with HER2 negative metastatic or locally advanced Metaplastic Breast Cancer (MpBC)

[0157] Study Description

[0158] This is a Phase II trial with a dose escalation / de-escalation lead-in that will investigate the efficacy and safety of an iNOS inhibitor and nab-paclitaxel in combination with alpelisib in patients with HER2 negative, metastatic or locally advanced MpBC. The prognosis for metastatic or locally advanced metaplastic breast cancer (MpBC), a rare and highly chemotherapy-resistant subtype of TNBC, is even worse than for non-metaplastic TNBC. with a median overall survival ranging from 3 to 8 months. Standard systemic chemotherapy remains the only available treatment option for patients with metastatic or locally advanced MpBC, even though this disease is largely refractory to cytotoxic drugs. Therefore, combination strategies to understand and overcome mechanisms of resistance to improve marginal chemotherapeutic efficacy are needed to improve the prognosis of these patients. It has been found that inducible nitric oxide synthase (iNOS) is a critical target for overcoming chemotherapy resistance in TNBC. iNOS expression is increased and associated with poor prognosis in invasive TNBC and MpBC. It has been shown that nitric oxide (NO) through iNOS induces many of the major oncogenic pathways such as RAS / ERK, HIFla, NF- KB, and others making this a unique oncogenic driver. One of the major oncogenic pathways activated by iNOS / NO is the phosphatidylinositol 3-kinase (PI3K) / Akt pathway. Alterations in the P13K / Akt pathway have also been linked with chemotherapy resistance, especially in MpBC. The data presented herein shows that the combinatory treatment of the PI3K inhibitor, alpelisib. to a pan-NOS inhibitor (L-NMMA) and nab-paclitaxel will increase objective response rate (ORR) in patients with HER2 negative metastatic or locally advanced MpBC. Both first- and second-line patients will be eligible for this trial. Patients may have received prior immunotherapy, per standard of care.

[0159] Trial Design and MethodDocket No. 38358.0004P1

[0160] The Phase II trial will investigate the efficacy and safety of a combination of an iNOS inhibitor, nab-paclitaxel and alpelisib in an estimated 36 patients (range 30-42), 18 years of age or older, with HER2 negative metastatic or locally advanced MpBC.

[0161] A total of 12 eligible patients have been enrolled to date. One patient was not eligible, and one was in screening at the time the annual report was submitted to the FDA. The age ranges were from 33 to 84 years old. Among the 11 known eligible patients, treatment duration ranged from one to nine cycles. Six patients discontinued treatment due to progressive disease and two due to toxicities. One withdrew from the trial after receiving nine cycles of therapy. The majority of the participants have metastatic MpBC.

[0162] Treatment

[0163] Patients in the study will receive L-NMMA (20 mg / kg intravenously [IV] over 2 hours days 15) and nab-paclitaxel (260 mg / m2IV day 1). In the dose escalation / de- escalation lead-in, alpelisib will be administered to the initial cohort of 3 patients at 250 mg po daily days 1-21 with escalation and de-escalation based on DLT determination as summarized in Table 7. In the phase II portion of the trial, patients will initiate alpelisib orally once daily days 1-21 at the recommended phase 2 dose identified in the escalation / de- escalation lead-in. Treatment will continue until progression, toxicity or patient decision to withdraw from study therapy. While on treatment patients will also receive amlodipine (10 mg orally once daily Days 0-5, aspirin (81 mg orally once daily), metformin (initiated at 500 mg orally once daily and escalated as needed) and cetirizine (10 mg orally once daily).

[0164] Initial enrollment included three patients at Dose Level 0 (alpelisib 250 mg on Days 1-21), with two patients experiencing dose-limiting toxicity. Subsequently, following the Bayesian Optimal Interval design, the dose was reduced to Level -1 (alpelisib 250 mg on Days 1-10) for the subsequent cohort. The Data Safety Monitoring Committee reviewed the dose-limiting toxicity' criteria and affirmed the necessity of dose reduction as per protocol requirements and to uphold regimen tolerability. From the first cohort of three patients treated at Dose Level -1. one patient experienced dose-limiting toxicity. Per Bayesian Optimal Interval design, the next cohort was to be treated at the same Dose Level -1. The second cohort of three patients treated at Dose Level -1 also had one patient experience dose-limiting toxicity.

[0165] The recommended phase 2 dose of alpelisib was determined at 250 mg (Days 1-10) based on the maximum tolerated identified using the Bayesian Optimal Interval design. This dose was expected to optimize therapeutic benefit while maintaining an acceptableDocket No. 38358.0004P1 safety profile. At this dose level, with a total of nine patients treated, four experienced doselimiting toxicities. Three of the four dose-limiting toxicities were rash events attributed to alpelisib. In the interest of patient safety and to reduce treatment-related toxicity, patients will be treated at Dose Level -2, which includes of 200 mg of alpelisib administered on Days 1- 21 of each cycle. The recommended phase 2 for the combination is administration of 20 mg / kg L-NMMA by 2-hour IV infusion Days 1-5, 260 mg / m2nab-paclitaxel Day 1, and 200 mg alpelisib Days 1-21 for each 21 -day cycle.

[0166] Alpelisib Supportive Medications

[0167] Metformin will be initiated at 500 mg once daily starting at one week prior to treatment. Based on tolerability and serial monitoring of blood sugar levels, metformin dose may be increased to 500 mg twice daily, followed by 500 mg with breakfast and 1000 mg with dinner, followed by further increase to 1000 mg twice daily if needed. Insulin should be used sparingly unless medically necessary for as brief of time as possible. Hyperglycemia should primarily be managed by holding and dose reductions of alpelisib along with escalation of metformin. Insulin sensitizers and / or SGLT2i will be used as second antidiabetic agents. For prophylaxis of alpelisib skin rash patients will be treated with cetirizine 10 mg daily.

[0168] L-NMMA Supportive Medications

[0169] Transient hypertension can frequently be observed while on therapy, and is ty pically a transient, clinically insignificant event, effectively managed by short term administration or intensification of pre-existing antihypertensive therapy. Amlodipine will be administered as 10 mg orally once daily for 6 days each cycle to reduce risk of sustained Grade 3 or Grade 4 hypertension. Amlodipine administration will start the evening before the first dose of L-NMMA and should be administered at least 30 minutes prior to the start of each L-NMMA infusion. Grade 3 hypertension lasting 72 hours or more will be considered a DLT. Amlodipine dose may be adjusted by the treating physician if needed. Amlodipine dose will be held if systolic blood pressure remains below 100 mmHg. Patients on chronic hypertensive therapy should add the amlodipine to their regular regimen for the 6 days. There may be a link between angi oedema and amlodipine therapy. All patients receiving amlodipine will be monitored for angioedema. Inhibition of NOS signaling may activate prostaglandin pathways, which may be abrogated safely by low-dose aspirin (81 mg daily).

[0170] Nab-paclitaxelDocket No. 38358.0004P1

[0171] 260 mg / m2will be administered IV on Day 1 of each cycle, every 3 weeks until progression, toxicity or patient withdraws from the study. The use of hematopoietic grow th factor support is permitted, as standard of care.

[0172] Table 7. Proposed Dose Levels

[0173] Efficacy Results

[0174] Of the nine patients evaluated for efficacy in the lead-in portion of the study the overall response rate (complete response+partial response) was 44.4% (4 of 9), while the clinical benefit rate (complete response+partial response+stable disease) w as 77.8% (7 / 9).

[0175] Safety Results

[0176] Grade >3 adverse events were reported in nine patients. Grade >3 adverse events were considered related to L-NMMA in two patients.

[0177] Since the start of the study, there have been four dose limiting toxicities. One patient experienced Grade 3 rash, which w as reported as probably related to alpelisib, with an associated Grade 2 fever unrelated to the study drugs. Another patient experienced Grade 3 oral mucositis, which was also probably related to alpelisib. The third and fourth doselimiting toxicities were also Grade 3 rashes, which were both reported as probably related to alpelisib.

[0178] Patients receiving 200 mg of alpelisib experienced lesser adverse events.

[0179] Two patients experienced Grade 3 hypertension, which were related to L- NMMA. Hypertension quickly resolved within an hour for both patients, and the events were not considered dose limiting toxicities. Other adverse events considered related to L-NMMA include a Grade 1 bradycardia and Grade 1 tachycardia in the same patient. The Grade 1 tachycardia is not expected for L-NMMA based on evidence from past patients; however,Docket No. 38358.0004P1 since the event occurred during infusion of L-NMMA it cannot be certain that it is not related.

[0180] Finally, two patients were hospitalized for Grade 3 sepsis that were both possibly related to nab-paclitaxel. These were not dose limiting toxicities as they occurred past the dose limiting toxicity window. Example 3: Phase II of inavolisib with iNOS Inhibitor and Nab-paclitaxel in Patients with HER2 negative metastatic or locally advanced Metaplastic Breast Cancer (MpBC)

[0181] Study Description

[0182] This is a Phase II trial with a dose escalation / de-escalation lead-in that will investigate the efficacy and safety of an iNOS inhibitor and nab-paclitaxel in combination with inavolisib in patients with HER2 negative, metastatic or locally advanced MpBC. Patients may have received prior immunotherapy, per standard of care.

[0183] Trial Design and Method

[0184] The Phase II trial will investigate the efficacy and safety of a combination of an iNOS inhibitor, nab-paclitaxel and inavolisib in 10-20 patients, 18 years of age or older, with HER2 negative metastatic or locally advanced MpBC.

[0185] No patients have been enrolled to date.

[0186] Treatment

[0187] Patients in the study will receive L-NMMA (20 mg / kg intravenously [IV] over 2 hours days 15) and nab-paclitaxel (260 mg / m2IV day 1). In the dose escalation / de- escalation lead-in, inavolisib will be administered to the initial cohort of 3 patients at 6 mg po daily days 1-21 with escalation and de-escalation based on DLT determination as summarized in Table 8. In the phase II portion of the trial, patients will initiate inavolisib orally once daily days 1-21 at the recommended phase 2 dose identified in the escalation / de- escalation lead-in. Treatment will continue until progression, toxicity or patient decision to withdraw from study therapy. While on treatment patients will also receive amlodipine (10 mg orally once daily Days 0-5, aspirin (81 mg orally once daily), metformin (initiated at 500 mg orally once daily and escalated as needed) and cetirizine (10 mg orally once daily).

[0188] Inavolisib Supportive Medications

[0189] Metformin wall be initiated at 500 mg once daily starting at one w eek prior to treatment. Based on tolerability and serial monitoring of blood sugar levels, metformin dose may be increased to 500 mg twice daily, followed by 500 mg with breakfast and 1000 mgDocket No. 38358.0004P1 wi th dinner, followed by further increase to 1000 mg twice daily if needed. Insulin should be used sparingly unless medically necessary for as brief of time as possible. Hyperglycemia should primarily be managed by holding and dose reductions of inavolisib along with escalation of metformin. Insulin sensitizers and / or SGLT2i will be used as second antidiabetic agents. For prophylaxis of inavolisib skin rash patients will be treated with cetirizine 10 mg daily.

[0190] L-NMMA Supportive Medications

[0191] Transient hypertension can frequently be observed while on therapy, and is typically a transient, clinically insignificant event, effectively managed by short term administration or intensification of pre-existing antihypertensive therapy. Amlodipine will be administered as 10 mg orally once daily for 6 days each cycle to reduce risk of sustained Grade 3 or Grade 4 hypertension. Amlodipine administration will start the evening before the first dose of L-NMMA and should be administered at least 30 minutes prior to the start of each L-NMMA infusion. Grade 3 hypertension lasting 72 hours or more will be considered a DLT. Amlodipine dose may be adjusted by the treating physician if needed. Amlodipine dose will be held if systolic blood pressure remains below 100 mmHg. Patients on chronic hypertensive therapy should add the amlodipine to their regular regimen for the 6 days. There may be a link between angi oedema and amlodipine therapy. All patients receiving amlodipine will be monitored for angioedema. Inhibition of NOS signaling may activate prostaglandin pathways, which may be abrogated safely by low-dose aspirin (81 mg daily).

[0192] Nab-paclitaxel

[0193] 260 mg / m2will be administered IV on Day 1 of each cycle, every 3 weeks until progression, toxicity or patient withdraws from the study. The use of hematopoietic growth factor support is permitted, as standard of care.

[0194] Table 8. Proposed Dose LevelsDocket No. 38358.0004P16. EquivalentsAlthough the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entireties.The discussion herein provides a better understanding of the nature of the problems confronting the art and should not be construed in any way as an admission as to prior art nor should the citation of any reference herein be construed as an admission that such reference constitutes “prior art” to the instant application.All references including patent applications and publications cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application w as specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

Docket No. 38358.0004P1CLAIMSWe claim:

1. A method of treating chemoresistant and / or metaplastic breast cancer in a human subject in need thereof, said method comprising: a. administering 15 to 25 mg / kg or 18 to 22 mg / kg or 20mg / kg of L-NMMA; b. administering a therapeutically effective amount of a taxane; and c. administering a therapeutically effective amount of a PI3K inhibitor.

2. The method of claim 1, wherein the PI3K inhibitor is alpelisib, buparlisib, taselisib, idelalisib, duvelisib, leniolisib, or copanlisib.

3. The method of any one of claims 1 to 2, wherein the PI3K inhibitor is administered every' day of a treatment cycle.

4. The method of any foregoing claims, wherein the PI3K inhibitor is administered orally.

5. A method of treating chemoresistant and / or metaplastic breast cancer in a human subject in need thereof, said method comprising: a. administering 15 to 25 mg / kg or 18 to 22 mg / kg or 20mg / kg of L-NMMA; b. administering a therapeutically effective amount of a taxane; and c. administering a therapeutically effective amount of an mTOR inhibitor.

6. The method of claims 5. wherein the mTOR inhibitor is an ATP-competitive mTOR kinase inhibitor.

7. The method of claim 6. wherein the mTOR inhibitor is torin-1, torin-2, vistusertib. AZD8055, MLN0128. PI-103. GDC-0980, INK126, WYE-354, WAY-600, and WYE-687, Ku-0063794, XL388, or PP244, PP30.Docket No. 38358.0004P18. The method of claims 5, wherein the mTOR inhibitor is rapamycin, everolimus, or temsirolumus.

9. A method of treating chemoresistant and / or metaplastic breast cancer in a human subject in need thereof said method comprising: a. administering 15 to 25 mg / kg or 18 to 22 mg / kg or 20mg / kg of L-NMMA; b. administering a therapeutically effective amount of a taxane; and c. administering a therapeutically effective amount of an AKT inhibitor.

10. The method of claim 9, wherein the AKT inhibitor is capivasertib, perifosine, MK-2206, uprosertib, ipatasertib, afuresertib, or miransertib.

11. The method of any foregoing claims, wherein L-NMMA is administered on days 1, 2, 3, 4, and 5 of a treatment cycle or at least 3, 4, or 5 days of the first 7 days of a treatment cycle.

12. The method of any foregoing claims, wherein the taxane is docetaxel, paclitaxel, nab-paclitaxel, larotaxel, or cabazitaxel.

13. The method of claim 12, wherein the taxane is nab-paclitaxel and the therapeutically effective amount is between 240 mg / m2to 280 mg / m2or 260 mg / m214. The method of claim 13, wherein nab-paclitaxel is administered on days 1, 8, and / or 15 or day 1 of a treatment cycle or administered once per week during a treatment cycle.

15. The method of claim 12, wherein the taxane is docetaxel and the therapeutically effective amount is 90 to 110 mg / m2or 100 mg / m2once per treatment cycle.

16. The method of any foregoing claims, wherein is L-NMMA and the taxane are administered intravenously.

17. The method of any of the foregoing claims, wherein the subject has a comorbidity.Docket No. 38358.0004P118. The method of any of the foregoing claims, wherein the co-morbidity is obesity (BMI>30 or >30% body fat for females or >25% body fat for males).

19. The method of any of the foregoing claims, wherein the breast cancer is further characterized as HER2 negative metaplastic breast cancer (MpBC) or locally advanced MpBC.

20. The method of any of the foregoing claims, wherein the breast cancer is further characterized as chemorefractory, locally advanced breast cancer (LABC), triple negative breast cancer (TNBC),21. The method of the foregoing claims, wherein the breast cancer is further characterized as: HER2 negative MpBC with squamous and / or sarcomatoid elements, including osseous, chondroid, or spindle morphology; TNBC with squamous and / or sarcomatoid elements, including osseous, chondroid, or spindle morphology; or locally advanced inoperable or metastatic MpBC with measurable disease.

22. The method of any of the foregoing claims, wherein the subject may have received prior immunotherapy.

23. The method of any of the foregoing claims, wherein the treatment cycle is between 14-30 days or 17 to 25 days or 20 to 23 days or 21 days.

24. The method of any of the foregoing claims, wherein the subject completes 3, 4, 5, 6, 7. 8, 9, or 10 treatment cycles.

25. The method of any of the foregoing claims, further comprising administering amlodipine at 10 mg orally once daily for 6 days each cycle or days 0 to 5 each cycle or at an amount and frequency effective to reduce risk of hypertension related to L-NMMA administration.

26. The method of any of the foregoing claims, further comprising administering aspirin at an amount effective to 81 mg once daily.

27. The method of any of the foregoing claims, further comprising administering metformin starting one week prior to a first treatment cycle at 500 mg to 2000 mg per day.Docket No. 38358.0004P128. The method of any of the foregoing claims, further comprising administering an effective amount of an antihistamine, such as cetirizine, daily as a prophylaxis for a skin rash; wherein, if cetirizine, the effective amount is at 10 mg per day.

29. The method of any of the foregoing claims, wherein the breast cancer is metaplastic.

30. The method of any of the foregoing claims, wherein the breast cancer is chemoresistant or wherein the patient has previously received a therapy comprising a chemotherapeutic, optionally wherein the chemotherapeutic is a taxane.

31. The method of any of the foregoing claims, wherein the subject has previously received an immunotherapy.

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