Carla Morales

Aurora A inhibitor (MLN8237) plus Vincristine plus Rituximab is synthetic lethal and a potential curative therapy in aggressive B-cell non-Hodgkin lymphoma

Purpose: Aurora A and B are oncogenic serine/threonine kinases that regulate mitosis. Overexpression of Auroras promotes resistance to microtubule-targeted agents. We investigated mechanistic synergy by inhibiting the mitotic spindle apparatus in the presence of MLN8237 [M], an Aurora A inhibitor with either vincristine [MV] or docetaxel [MD] in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The addition of rituximab [R] to MV or MD was evaluated for synthetic lethality. Experimental Design: Aggressive B-NHL cell subtypes were evaluated in vitro and in vivo for target modulation and anti-NHL activity with single agents, doublets, and triplets by analyzing cell proliferation, apoptosis, tumor growth, survival, and mechanisms of response/relapse by gene expression profiling with protein validation. Results: MV is synergistic whereas MD is additive for cell proliferation inhibition in B-NHL cell culture models. Addition of rituximab to MV is superior to MD, but both significantly induce apoptosis compared with doublet therapy. Mouse xenograft models of mantle cell lymphoma showed modest single-agent activity for MLN8237, rituximab, docetaxel, and vincristine with tumor growth inhibition (TGI) of approximately 10% to 15%. Of the doublets, MV caused tumor regression, whereas TGI was observed with MD (approximately 55%–60%) and MR (approximately 25%–50%), respectively. Although MV caused tumor regression, mice relapsed 20 days after stopping therapy. In contrast, MVR was curative, whereas MDR led to TGI of approximately 85%. Proliferation cell nuclear antigen, Aurora B, cyclin B1, cyclin D1, and Bcl-2 proteins of harvested tumors confirmed response and resistance to therapy. Conclusions: Addition of rituximab to MV is a novel therapeutic strategy for aggressive B-NHL and warrants clinical trial evaluation. Clin Cancer Res; 18(8); 2210–9. ©2012 AACR.

Alisertib Added to Rituximab and Vincristine Is Synthetic Lethal and Potentially Curative in Mice with Aggressive DLBCL Co-Overexpressing MYC and BCL2

Pearson correlation coefficient for expression analysis of the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) demonstrated Aurora A and B are highly correlated with MYC in DLBCL and mantle cell lymphoma (MCL), while both Auroras correlate with BCL2 only in DLBCL. Auroras are up-regulated by MYC dysregulation with associated aneuploidy and resistance to microtubule targeted agents such as vincristine. Myc and Bcl2 are differentially expressed in U-2932, TMD-8, OCI-Ly10 and Granta-519, but only U-2932 cells over-express mutated p53. Alisertib [MLN8237 or M], a highly selective small molecule inhibitor of Aurora A kinase, was synergistic with vincristine [VCR] and rituximab [R] for inhibition of cell proliferation, abrogation of cell cycle checkpoints and enhanced apoptosis versus single agent or doublet therapy. A DLBCL (U-2932) mouse model showed tumor growth inhibition (TGI) of ∼10–20% (p = 0.001) for M, VCR and M-VCR respectively, while R alone showed ∼50% TGI (p = 0.001). M-R and VCR-R led to tumor regression [TR], but relapsed 10 days after discontinuing therapy. In contrast, M-VCR-R demonstrated TR with no relapse >40 days after stopping therapy with a Kaplan-Meier survival of 100%. Genes that are modulated by M-VCR-R (CENP-C, Auroras) play a role in centromere-kinetochore function in an attempt to maintain mitosis in the presence of synthetic lethality. Together, our data suggest that the interaction between alisertib plus VCR plus rituximab is synergistic and synthetic lethal in Myc and Bcl-2 co-expressing DLBCL. Alisertib plus vincristine plus rituximab [M-VCR-R] may represent a new strategy for DLBCL therapy.

Reciprocal feedback inhibition of the androgen receptor and PI3K as a novel therapy for castrate-sensitive and -resistant prostate cancer

Gain-of-function of the androgen receptor (AR) and activation of PI3K/AKT/mTOR pathway have been demonstrated to correlate with progression to castration-resistant prostate cancer (CRPC). However, inhibition of AR or PI3K/mTOR alone results in a reciprocal feedback activation. Therefore, we hypothesized that dual inhibition of the AR and PI3K/mTOR pathway might lead to a synergistic inhibition of cell growth and overcome drug resistance in CRPC. Here, we reported that androgen-depletion increased AR protein level and Akt phosphorylation at Ser473 and Thr308 in LNCaP cells. Moreover, we developed resistance cell lines of LNCaP to Enzalutamide (or MDV3100), an AR inhibitor (named as LNCaP ‘MDV-R’) and PF-04691502, a PI3K/mTOR inhibitor (named as LNCaP ‘PF-R’). MTS analysis showed that LNCaP ‘PF-R’ was strongly resistant to Enzalutamide treatment, and on the other hand, LNCaP ‘MDV-R’ was 6-fold resistant to PF-04691502 treatment. Mechanistically, LNCaP ‘MDV-R’ cells had significantly reduced AR, loss of PSA and increase Akt activity in contrast with LNCaP ‘PF-R’ cells. Combined inhibition of PI3K/mTOR and AR pathways with a variety of small molecular inhibitors led to a synergistic suppression of cell proliferation and a profound increase of apoptosis and cell cycle arrest in both androgen-dependent LNCaP and independent CRPC 22Rv1 cell lines. In conclusion, this study provides preclinical proof-of-concept that the combination of a PI3K/mTOR inhibitor with an AR inhibitor results in a synergistic anti-tumor response in non-CRPC and CRPC models.

Disruption of Aneuploidy and Senescence Induced by Aurora Inhibition Promotes Intrinsic Apoptosis in Double Hit or Double Expressor Diffuse Large B-cell Lymphomas

Double hit (DH) or double expressor (DE) diffuse large B-cell lymphomas (DLBCL) are aggressive non-Hodgkins lymphomas (NHL) with translocations and/or overexpressions of MYC and BCL-2, which are difficult to treat. Aurora kinase (AK) inhibition with alisertib in DH/DE-DLBCL induces cell death in ∼30%, while ∼70% are aneuploid and senescent cells (AASC), a mitotic escape mechanism contributing to drug resistance. These AASCs elaborated a high metabolic rate by increased AKT/mTOR and ERK/MAPK activity via BTK signaling through the chronic active B-cell receptor (BCR) pathway. Combinations of alisertib + ibrutinib or alisertib + ibrutinib + rituximab significantly reduced AASCs with enhanced intrinsic cell death. Inhibition of AK + BTK reduced phosphorylation of AKT/mTOR and ERK-1/2, upregulated phospho-H2A-X and Chk-2 (DNA damage), reduced Bcl-6, and decreased Bcl-2 and Bcl-xL and induced apoptosis by PARP cleavage. In a DE-DLBCL SCID mouse xenograft model, ibrutinib alone was inactive, while alisertib + ibrutinib was additive with a tumor growth inhibition (TGI) rate of ∼25%. However, TGI for ibrutinib + rituximab was ∼50% to 60%. In contrast, triple therapy showed a TGI rate of >90%. Kaplan–Meier survival analysis showed that 67% of mice were alive at day 89 with triple therapy versus 20% with ibrutinib + rituximab. All treatments were well tolerated with no changes in body weights. A novel triple therapy consisting of alisertib + ibrutinib + rituximab inhibits AASCs induced by AK inhibition in DH/DE-DLBCL leading to a significant antiproliferative signal, enhanced intrinsic apoptosis and may be of therapeutic potential in these lymphomas. Mol Cancer Ther; 16(10); 2083–93. ©2017 AACR.

SF1126, a Pan-PI3K Inhibitor has Potent Pre-Clinical Activity in Aggressive B-Cell Non-Hodgkin Lymphomas by Inducing Cell Cycle Arrest and Apoptosis

The PI3K pathway is activated in a variety of human tumors including B-cell Non Hodgkin Lymphoma (B-NHL). Targeting this pathway has been validated in solid tumors, leukemia and lymphomas. SF1126, a novel pan-PI3K inhibitor designed by conjugating RGD peptide to LY294002 facilitates clinical testing of this prodrug, suppresses growth and survival of multiple solid tumors in vitro and in vivo. In this study, we demonstrated SF1126 had potent activity in a panel of aggressive B-NHL cell lines. Cells treated with SF1126 exhibited decreased phosphorylation of Akt and GSK-3β confirming the mechanism of action of a PI3K inhibitor. Also, treatment of B-NHL cell lines with SF1126 induced apoptosis in a dose-dependent manner and inhibited cell proliferation with an IC50 < 4 μM. However, the selective p110δ inhibitor, CAL-101 was less potent in inducing apoptosis and inhibiting cell proliferation compared to SF1126. Moreover, SF1126 induced G1 cell cycle arrest significantly at low concentrations which contributed to suppression of cell proliferation and corroborated to a decrease in cyclin D1. Finally, rituximab enhanced apoptosis induced by SF1126 and CAL-101. Taken together, our findings provide for the first time that SF1126 inhibits the constitutively activated PI3K/Akt pathway in aggressive B-cell NHL cell lines with associated inhibition of cell cycle progression, cell proliferation and promotion of apoptosis. These findings suggest that SF1126 is a novel therapeutic strategy in aggressive B-cell NHL and warrants early phase clinical trial evaluation ± rituximab.

Co-targeting aurora kinase with PD-L1 and PI3K abrogates immune checkpoint mediated proliferation in peripheral T-cell lymphoma: a novel therapeutic strategy

Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate ∼30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4+/CD8+ cells, we hypothesized that Program Death Ligand-1 (PD-L1) expression is essential for uncontrolled proliferation. Combination of alisertib with PI3Kα (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (∼9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-κB expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) ∼30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI >90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.

Abstract 2578: Synthetic lethal approaches to aurora inhibition in aggressive B-cell non-Hodgkin lymphoma

We explored synthetic lethality in the context of aurora inhibition with novel combination therapies for aggressive B-NHL. Aurora A and B are serine/threonine kinases and when amplified dysregulate the mitotic phase of the cell cycle. Pre-clinical and clinical evidence demonstrate that aurora inhibition has therapeutic benefit in aggressive B-NHL. Alisertib, an aurora inhibitor has single agent activity in B-NHL mouse xenograft tumors and clinical trials. Pre-clinical studies demonstrate Bruton9s tyrosine kinase (BTK) is persistently expressed despite alisertib therapy. TMAs for DLBCL (S0313, S0515, N = 60) and MCL (S0213, S0601, N = 45) were generated to evaluate aurora A, B and BTK expression by IHC. We developed B-NHL cell lines resistant to alisertib to evaluate mechanisms of acute and chronic resistance and knock down of aurora A and B to evaluate Myc, Bcl2 and BTK expression. Mouse B-NHL xenograft models were conducted to demonstrate synthetic lethality of aurora plus BTK inhibition. We demonstrate synergistic inhibition of cell proliferation associated with apoptosis, particularly in ABC and double hit DLBCL and mantle cell lymphoma (MCL) cells. Ibrutinib completely inhibited BTK and AKT activity at 3.0μM without affecting BTK or AKT protein level in B-NHL cells that co-over-express Myc and Bcl-2. Aurora A and B knockdown show compromised oncogene expression. IHC indicated in DLBCL and MCL aurora B to be differentially over-expressed versus aurora A and BTK. Alisertib plus ibrutinib was highly synergistic in inhibiting cell proliferation and inducing apoptosis in B-NHL cells. Moreover, this effect was amplified with the addition of rituximab. Acute and chronic resistance to alisertib was due to ERK1/2 activation. Significantly greater apoptosis was induced in the triplet combination (alisertib + ibrutinib + rituximab) compared to doublet combinations (ibrutinib + rituximab or alisertib + ibrutinib or alisertib + rituximab). Mouse xenograft models of MCL (Granta-519) and double hit DLBCL (U2932) showed ibrutinib alone had no anti-B-NHL activity. Ibrutinib + alisertib lead to tumor growth inhibition (TGI) of ∼50% in MCL and ∼30% in DLBCL (p = 0.005). Interestingly, ibrutinib + rituximab lead to a TGI of ∼60% (p = 0.005) in both B-NHL mouse models. In contrast, triplet therapy with alisertib plus ibrutinib plus rituximab demonstrated a statistically significant tumor TGI of ∼70-90% (p = 0.005) in both B-NHL mouse models with an associated enhanced overall survival (>70 days). Harvested tumors at the end of treatment(s) demonstrated target inhibition and increased apoptosis with triple therapy. Inhibition of BTK plus rituximab in the presence of aurora inhibition is synthetic lethal in B-NHL and warrant clinical trial evaluation. Citation Format: Daruka Mahadevan, Carla Morales, Laurence Cooke, Manjari Pandey, Catherine Spier, Wenqing Qi. Synthetic lethal approaches to aurora inhibition in aggressive B-cell non-Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2578. doi:10.1158/1538-7445.AM2015-2578

Abstract 675: Dual inhibition of the androgen receptor and PI-3K/mTor pathways has significant antitumor activity in castrate-resistant prostate cancer

Inhibition of the androgen receptor (AR) results in activation of the PI-3K/mTOR pathway in PTEN deficient prostate cancer cells by reciprocal feedback inhibition (Carver BS, 2011). Enzalutamide or PI-3K/mTor inhibition result in feedback activation and combined inhibition abrogated this effect. We explored AR inhibition in CRPC cell lines with low, intermediate and high AR expression in the context of PI-3K/mTor inhibition to identify biomarkers of response and resistance. We utilized no/low (DU145, PC3), intermediate (LNCaP) and high (22Rv1,VCap) AR expressing prostate cancer (PC) cell lines to evaluate anti-tumor activity with 4 pan-PI3K/mTOR inhibitors (GDC-0980, GDC-0941, LY294002, PF-04691502), enzalutamide and abiraterone as single agents or various in combinations at clinically relevant doses. Cytotoxic activity of enzalutamide was more potent than abiraterone (IC 50 9s was 10µM) in LNCaP cells, however, the opposite was seen in DU145 and PC-3 cell lines. High AR expressing cells (22Rv1, VCap) were more sensitive to AR inhibition. PI-3K/mTor SMIs GDC-0980, GDC-0941 or PF-04691502 had IC 50 9s in the range 50-500nM while LY294002 had an IC 50 16-24 µM except for DU145. This cell line had increased IC 50 values comparatively for PI-3K/mTor SMIs GDC-0980, GDC-0941 or PF-04691502 (1.5-3.75 µM) and LY29002 (48.5 µM). The combination index (CI) isobologram method indicated significant synergism for PI-3K/mTOR SMIs plus enzalutamide or abiraterone in all PC cell lines irrespective of AR status. Combination treatments were associated with increased apoptosis (Annexin-V staining and PARP-cleavage assay) in LNCaP and VCaP > 22Rv1 > DU145 and PC3. Cell cycle analysis by flow cytometry demonstrated that enzalutamide induces G1 arrest in LNCaP and DU145 cells and G2/M arrest in PC3 cells. PI-3K inhibition induces S arrest in LNCaP and DU145 and G2/M arrest in PC3 cells. The combination of enzalutamide plus PI-3K inhibition enhanced S arrest in LNCaP while in DU145 and PC3, G0/G1 arrest was prominent. PI-3K SMI plus enzalutamide completely inhibited pAkt308/473, pS6 and p4EBP1 in LNCaP cells, pAkt473 in PC3 cells and pS6 and p4EBP1 in 22Rv1 cells. Enzalutamide alone or in combination with PI-3K inhibition also suppressed AR expression in 22Rv1 cells. A 22Rv1 mouse xenograft model is evaluating safety and efficacy of the combination. This is a novel therapeutic strategy for CRPC patients to be evaluated in a clinical trial. Citation Format: Daruka Mahadevan, Carla Morales, Laurence S. Cooke, Bradley Somer, Wenqing Qi. Dual inhibition of the androgen receptor and PI-3K/mTor pathways has significant antitumor activity in castrate-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 675. doi:10.1158/1538-7445.AM2014-675

Abstract 3388: Novel aurora plus Bruton's tyrosine kinase-targeted therapies for aggressive B-cell non-Hodgkin's lymphoma.

Exploring synthetic lethality in the context of signaling pathways with novel targeted agents may yield effective combination therapies for aggressive B-NHL. Auroras are a family of mitotic oncogenic serine/threonine kinases intimately involved in high fidelity regulation of cell division. We previously demonstrated that aberrant Aurora (A and B) expression portends a poor survival in mantle cell lymphoma (MCL) patients. Aberrant aurora expression leads to genetic instability, polyploidy, and resistance to microtubule targeted agents (MTAs). We demonstrated that combination of an Aurora inhibitor MLN8237 (alisertib) or AT9283 with MTAs (e.g. taxol or vincas) leads to a mechanistic decoupling of the mitotic spindle manifesting as a synergistic apoptotic response in cell culture and in mouse xenograft models of aggressive B-NHL. Gene expression profiling of harvested mouse xenograft tumors at the end of treatment (3 weeks) with an aurora inhibitor + MTA showed over-expression of several markers of resistance of which BTK (Bruton9s tyrosine kinase) was the most prominent. BTK plays a role in uncontrolled activation and proliferation of malignant B-cells via the chronic active B-cell receptor pathway in aggressive B-NHL. We hypothesized that targeting proliferation [BTK] and replication [Aurora] would be a novel therapeutic strategy in aggressive B-NHL. We demonstrate that both aurora (A and B) and BTK are highly over-expressed in B-NHL cell lines (Granta-519, SUDHL4, SUDHL10, OCI-Ly-10, TMD8, U-2932) and harvested mouse xenograft tumour treated with an Aurora inhibitor + vincristine and in patient samples. IC 50 values for AT9283 is 5-10nM, and CNX652 is 0.07-5μM. The combination index (CI) indicated significant synergism for AT9283 plus CNX-652 or vice versa. Combination therapy showed increased apoptosis (flow cytometry, PARP-cleavage) in a dose-dependent manner. Both AT9283 and AVL-292 (CNX-652 is a structural analog with similar in vitro and in vivo properties to AVL-292) are in early phase clinical trials in hematologic malignancies. We utilized clinically relevant doses to evaluate the efficacy of single agent and combination therapy of AT9283 and CNX652 in mouse xenograft models (Granta-519) of MCL. The first mouse model evaluated single agent dose-response and target modulation by Western blotting (pHisH3, pBTK) in tumors harvested at the end of 3 weeks of treatment (CNX-652 daily oral for 3 weeks or AT9283 daily IV for 3 weeks, n=12 mice per cohort). The second study evaluated the combination of AT9283 and CNX-652 for endpoints of response, overall survival and target modulation. Gene expression and protein analysis of harvested tumors at the end of treatment (3 weeks) have been interrogated to ascertain the mechanistic role of Aurora plus BTK inhibition. The combination of AT9283 + CNX652 represents a novel therapeutic strategy for aggressive B-NHL [Funded by the Hope Foundation]. Citation Format: Daruka Mahadevan, Carla Morales, Russell Karp, Laurence Cooke, John Lyons, Juswinder Singh, Wenqing Qi. Novel aurora plus Bruton9s tyrosine kinase-targeted therapies for aggressive B-cell non-Hodgkin9s lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3388. doi:10.1158/1538-7445.AM2013-3388