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. 2022 Mar 30;13(4):615–622. doi: 10.1021/acsmedchemlett.1c00673

Azapodophyllotoxin Causes Lymphoma and Kidney Cancer Regression by Disrupting Tubulin and Monoglycerols

Arvin M Gouw †, Vineet Kumar ‡, Angel Resendez ‡, Fidelia B Alvina †, Natalie S Liu †, Katherine Margulis §, Ling Tong †, Richard N Zare §, Sanjay V Malhotra ‡,∥,⊥,*, Dean W Felsher †,*
PMCID: PMC9014495  PMID: 35450373

Abstract

graphic file with name ml1c00673_0005.jpg

A natural compound screen identified several anticancer compounds, among which azapodophyllotoxin (AZP) was found to be the most potent. AZP caused decreased viability of both mouse and human lymphoma and renal cell cancer (RCC) tumor-derived cell lines. Novel AZP derivatives were synthesized and screened identifying compound NSC750212 to inhibit the growth of both lymphoma and RCC both in vitro and in vivo. A nanoimmunoassay was used to assess the NSC750212 mode of action in vivo. On the basis of the structure of AZP and its mode of action, AZP disrupts tubulin polymerization. Through desorption electrospray ionization mass spectrometry imaging, NSC750212 was found to inhibit lipid metabolism. NSC750212 suppresses monoglycerol metabolism depleting lipids and thereby inhibits tumor growth. The dual mode of tubulin polymerization disruption and monoglycerol metabolism inhibition makes NSC750212 a potent small molecule against lymphoma and RCC.

Keywords: Azapodophyllotoxin, Kidney Cancer, Lymphoma, Monoglycerol, Tubulin

Lymphoma1 and renal cell cancer (RCC)2 are common malignancies. Advanced kidney cancer is incurable. Although a multitude of treatments exist for lymphoma, many patients succumb to their disease.3

Microtubules are cytoskeletal protein polymers composed of α- and β-tubulin heterodimers. They are involved in essential cellular processes such as migration, intracellular transport, and mitosis.4 Inhibition of microtubule polymerization leads to disruption of mitotic spindle formation, blocks mitosis, and arrests the cell cycle in the G2/M phase, leading to apoptosis. Thus, tubulin is considered to be an important target for anticancer drug development. Most of the drugs that inhibit microtubule assembly, including paclitaxel (taxane), vinblastine, and vincristine (vinca alkaloids), and podophyllotoxin (lignans) are derived from natural products.5,6 These drugs, despite their significant clinical relevance, possess serious problems in terms of pharmacokinetics, toxicity, and resistance that limit their therapeutic potential.7,8 Also, because of the presence of complex ring systems and multiple chiral centers, their structures are very complex, which require difficult and laborious synthetic steps. This makes a large-scale supply of these compounds challenging and impedes lead optimization. Thus, there is much interest in the development of new and structurally simple microtubule-binding anticancer agents that can be synthesized easily to overcome these limitations. Even if these novel compounds might have moderated cytotoxic potencies compared with the parent structurally complex natural product, the ease of preparation of carefully designed libraries of analogues would lead to more informative structure activity relationship (SAR) studies and expeditious structure optimization.

In the last two decades, considerable efforts have been made to synthesize aza analogues of podophyllotoxin, such as azapodophyllotoxins (AZPs), with heteroatoms at different positions of the podophyllotoxin skeleton, while keeping the basic podophyllotoxin structure.9 The use of simple one-step multicomponent reactions (MCRs) has greatly increased the speed at which AZPs can be synthesized, making available diverse analogues to test against a diverse population of carcinomas and other diseases. Often, several AZPs retain a great fraction of the cytotoxicity associated with the parent lignan. We have previously reported the synthesis of novel AZPs and their screening data on National Cancer Institute’s 60 human tumor cell lines (NCI-60).9 Some of these AZPs showed highly potent activity against a variety of cancer cell lines, including leukemia and renal cancer. Here, we present new analogues of these AZP compounds and demonstrate their anticancer activities in various leukemia and renal cancer cell lines. We explored the in vivo efficacy and in-depth the mechanism of actions of AZPs as the inhibitors of tubulin polymerization using our lead compound NSC750212.

To quantifiably compare the effects of NSC750212 at the protein level and monitor changes in the proteomic landscape during treatment, we used a nanofluidic proteomic immunoassay (NIA).10,11 This therapeutic monitoring technique is a highly sensitive method for measuring protein expression in clinical samples. Here, we used NIA to analyze fine needle aspirates (FNA) and core biopsies of transplanted xenografts. This helps to monitor changes in tubulin protein levels throughout the course of treatment of mice with NSC750212 and determine whether there is a significant quantifiable difference between the treatment group in comparison with the control.

To determine effects of NSC750212 on cancer metabolism, we utilized ambient ionization mass spectrometry, which is a collective term describing all mass spectrometric ionization methods that are capable of ionizing the constituents of natural samples under ambient conditions.12 In this regard, ambient mass spectrometry (MS) methods are well-suited for studying tissue samples, without having to use any chemical modification, ideally in vivo, giving outstanding significance to these methods in the field of cancer research. The first ambient MS method described was desorption electrospray ionization mass spectrometry (DESI-MS), which was implemented by directing a pneumatically assisted solvent electrospray onto the surface of interest. We have utilized DESI-MS to detect metabolomic changes in our mouse models.13 However, we will now utilize DESI-MSI to uncover the mechanism by which NSC750212 disrupts cancer progression metabolically.

Podophyllotoxin (A) (Figure 1), a lignan obtained from plants of the Podophyllum genus, is an important ligand with remarkable microtubule assembly inhibitory activity. However, its therapeutic use has been restricted due to its high toxicity.14 Extensive efforts to reduce its toxic effects led to the development of its semisynthetic derivatives etoposide and teniposide, which are currently used in combination cancer chemotherapy but have different mechanisms of action than podophyllotoxin.15,16 Reports on completely synthetic analogues of podophyllotoxins are rather limited due to its highly complex structure. Recently, there has been a growing interest in the development of aza analogues of A that retain key structural aspects and can be synthesized in a single-step, one-pot multicomponent reaction.17 Earlier, it has been demonstrated that simplified AZPs, such as 4-aza-2,3-didehydropodophyllotoxins, retain the cytotoxic activity of the parent compound A.18 Moreover, the stereocenters at C-2 and C-3 have been removed in these compounds. Hence, there is no possibility of epimerization at the C-2 center, which has hampered the clinical development of A and its complex derivatives due to the formation of significantly less potent cis-lactone metabolite.19 On the basis of these observations, we developed novel AZP derivatives (Figure 1), which showed remarkable anticancer activities in various NCI-60 human tumor cell lines including leukemia and renal cancer. These compounds contain only one stereocenter, making them structurally simpler. They were synthesized in a single step in good yield from commercially available starting materials.

Figure 1.

Figure 1

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Structures of AZP derivatives used in current studies showing (A) podophyllotoxin, showing modifications (B) for NSC750212, NSC750719, and AR-02; (C) for AR-038 and AR-061; (D) NSC750722, NSC756089, AR-03, AR-051, and AR-065.

We reexamined the hit compounds from our previous study of NSC750212, NSC750719, NSC750722, and NSC756089 on our MYC-driven RCC line derived from our transgenic mouse model using a smaller dose range (280 nM, 140 nM, and 70 nM).11 All these compounds had high sensitivity (Figure S1A). Even at 70 nM, NSC750212 causes ∼90% reduction of cell proliferation compared to the vehicle control (dimethyl sulfoxide (DMSO)). At 140 nM, NSC750212 causes a twofold suppression of proliferation when compared to 70 nM, and any higher concentration (280 nM) shows no further efficacy. NSC756089 was the least sensitive of all four compounds.

To expand the SAR, six additional analogues of these AZP derivatives were synthesized, namely, AR-02, AR-03, AR-038, AR-051, AR-061, and AR-065, with different substituents on the C-1 phenyl ring (Figure 1). The AZP compounds showed efficacy in a murine tumor-derived RCC line. Next, we examined the efficacy of NSC750212 and newly synthesized analogues in the human RCC tumor-derived cell line A498 (Figure 2A). At 70 nM, the two compounds with no substitution on the C-1 phenyl ring, AR-02 and AR-03, had less efficacy (30% reduction in viability) compared to NSC750212 (95% suppression). Compounds AR-061 and AR-065 had 83% and 80% decrease in viability, respectively, comparable to that of NSC750212. The most potent of the newly synthesized compounds are AR38–1 and AR51–1, both having a single bromine substitution on the C-1 phenyl ring, with 90% and 87% suppression, respectively. However, NSC750212 is still the most potent compound.

Figure 2.

Figure 2

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(A) From left to right: control, AR-02, AR-03, AR-038, AR-051, AR-061, AR-065, and NSC750212 efficacy in the suppression of proliferation in vitro in the human RCC A498 cell line over the course of 48 h. Statistical significance by t-test for AZP analogues compared to control, **p < 0.01, n.s. = not significant. (B) From left to right: human lymphoma lines CCRF, DND-41, KOPT, and MYC-driven lymphoma lines P493–6 and 6780 proliferative responses to 70, 140, or 280 nM increases in dosage of NSC750212. Statistical significance by t-test for NSC750212 of different concentrations compared to DMSO, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant. (C) Comparison of xenograft volume between the DMSO-treated group and the NSC750212-treated group upon termination of treatment in RCC xenografts. Statistical significance by t-test for NSC750212 compared to control, n.s. = not significant. (D) Comparison of xenograft volume between the DMSO-treated group and the NSC750212-treated group upon termination of treatment in lymphoma xenografts. Statistical significance by t-test for NSC750212 compared to control, **p < 0.01.

The NCI screen showed positive results for renal cell carcinoma and lymphomas. NSC750212 showed a dose-dependent suppression of proliferation in various human lymphoma lines of CCRF (T lymphoblast), DND-41 (a T acute lymphoblastic leukemia (T-ALL), and KOPT-K1 (T-ALL) as well as in MYC-driven murine tumor-derived lymphoma lines1 (Figure 2B). At 70 nM concentration, it reduced proliferation by 90% in the CCRF and 6780 cell lines, 95% in DND-41, 85% in KOPT and P-493. At 140 nM, proliferation decreased by 92% and 94% in CCRF and 6780. But, DND-41 exhibited no dose response, possibly due to 70 nM being the maximally effective dose in this cell line. KOPT exhibited 87% inhibition of proliferation. P-493 exhibited a 90% reduction in proliferation. At 280 nM concentration, maximally inhibited 6780 and CCRF and P-493 showed an inhibition of proliferation of 96% and 98%, respectively. KOPT increased to ∼89% reduction of proliferation. NSC750212 displays a dose-dependent response in CCRF, KOPT, P-493, and 6780, while reaching maximal dose in DND-41 at 70 nM, indicating a much higher efficacy than what the NCI screen indicates (Figure 2B), which is also the case for RCC (Figures S1A & S2A).

We measured to see whether NSC750212 impeded cancer growth in vivo. Since NSC750212 had the highest efficacy in vitro against the human RCC A498 line (Figure 2A) and the human lymphoma line CCRF (Figure 2B), we tested and found that NSC750212 decreased in vivo tumor growth of both human tumors after an intratumoral treatment for a week (Figure 2C,D).

The structure of NSC750212 combined with computational docking studies of azapodophyllotoxins suggested they may target tubulins. Using EMD Millipore Tubulin Polymerization Assay, NSC750212, Paclitaxel (negative control) and Nocodazole (positive control) were assays for the inhibition of tubulin polymerization. NSC750212 inhibited tubulin polymerization (Figure 3A). To examine in vivo, NIA was used to show that tubulin decreased as measured in fine needle aspirates (FNAs) of transplanted tumors after a treatment with NSC750212 (Figure 3B) by at least twofold. A size blot depiction of NIA also shows dramatic reduction of tubulin levels in the NSC750212-treated group (Figure 3C). There are no other charged isoforms of tubulin detected by NIA (Figure S2). This suggests that NSC750212 inhibits tubulin polymerization.

Figure 3.

Figure 3

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(A) (top curve) Paclitaxel as a tubulin polymerization stabilizer (negative control), (middle curve) Nocodazole as a tubulin polymerization destabilizer (positive control), and (bottom curve) NSC750212, which shows an even higher efficacy than the positive control. (B) (left to right) Control and NSC750212 FNAs, and control and NSC750212 core biopsies from mouse tumors taken during the course of treatment. (C) NIA data shown in gel display (similar to the western blot). (left to right) Control and NSC750212-treated ex vivo FNAs, and control and NSC750212-treated ex vivo core biopsies.

Upon the basis of our prior work, we speculated that AZPs may influence lipid metabolism.9,13 We examined the mechanism of action through an examination of lipid metabolism by DESI-MSI. The NSC750212 treatment increased monoglycerols (Figure 4). Previous work suggests that breakdown of monoglycerols is required for tumor growth.20 We infer that NSC750212 may have more potent anticancer activity because, in addition to their inhibition of tubulin, which has been known in cancer drug development,21−24 they affect lipid metabolism, which is also key for cancer metabolism.25−29

Figure 4.

Figure 4

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Comparison of DESI-MSI profiles of monoglycerols vs fatty acids in normal vs NSC750212-treated group upon termination of treatment in human lymphoma line, CCRF.

Here we have identified AZP derivatives with potent anti-neoplastic activity against RCC and lymphoma. Further, through a novel nanofluidic proteomic method, NIA,30 and in situ mass spectrometry method, DESI-MSI,31−34 we have shown that the most potent derivative, NSC750212, has both antitubulin activity and suppresses monoglycerol production. Hence, we have identified a modified natural product with potent anticancer activity with a novel mechanism of action.

Natural products35−38 have been a major source of pharmaceutical development due to their intrinsic biological relevance and have often been referred to as privileged structures that are likely to possess multiple biological activities. However, their complex structure and difficult synthesis impedes the hit-to-lead generation process of natural product-based drugs. One such example is podophyllotoxin,39−41 which is a potent but highly toxic tubulin polymerization inhibitor. We used a single-step, multicomponent reaction (MCR) to synthesize structurally simple aza analogues of podophyllotoxin referred to here as AZPs. The screening of our previously reported and newly synthesized AZP derivatives showed high efficacy in both murine and human RCC cell lines E28 and A498 (Figures S1A and Figure 2A). An SAR analysis suggests that compounds with an unsubstituted C-4 phenyl ring were the least active (AR-02 and AR-03) and that the ones with a trimethoxy substitution on the C-4 phenyl ring were the most active (NSC750212). Compounds with a halogenated substitution at the C-4 phenyl ring also showed promising activity (AR-038 and AR-051). The hit compound NSC750212 also showed dose-dependent inhibition in human lymphoma lines CCRF, DND-41, and KOPT and MYC-driven lymphoma lines P493–6 and 6780 (Figure 2B).

Our study demonstrates the importance of developing technologies to be able to assess the drug mechanism of action as well as monitoring its therapeutic response for both NIA and DESI-MSI. NIA has been previously used to detect the tissue origin of certain cancers as well as the driving oncogene of the cancer from a very small amount of sampling.8−10 Here we have developed NIA further to be able to make those measurements from preclinical studies to allow time course measurements of tumors in vivo. Moreover, this method can then be used to monitor the therapeutic response of a certain drug by measuring key biomarkers to assess the novel drug’s potency not only at the study’s end point. In addition to the mechanism of NSC750212 as a tubulin disruptor, DESI-MSI has uncovered another novel mechanism of NSC750212, through the inhibition of monoglycerol metabolism. Our results suggest that the high potency of NSC750212 may reflect this dual mechanism of action.

We report that the modification of a natural compound, AZP, accompanied by nanoproteomic and mass spectrometry methods that are instrumental in identifying that NSC750212 constitutes a novel tubulin and monoglycerol metabolic inhibitor with potent activity against RCC and lymphoma.

Human T-cell acute lymphoblastic leukemias CCRF, DND-41, KOPT, human B-cell Burkitt’s lymphoma P493–6, and murine lymphoma 6780 were used in this study and supplemented by RPMI media from Gibco, while the murine renal cell carcinoma E28 was supplemented by Dulbecco’s Modified Eagle’s Medium (DMEM); for further details see the Supporting Information. Cell counts were done using a hemocytometer.

The human T-ALL cell line CCRF was transplanted subcutaneously into immunodeficient NOD-scid gamma mice (NSG), which lack mature B-cells, T-cells, and natural killer cells. Prior to the transplantation, CCRF was cultured in RPMI supplemented with 10% (v/v) fetal bovine serum (FBS), 1% glutamine, 1% sodium pyruvate, 1% nonessential amino acids, and Antibiotic-Antimycotic. On the day of transplantation, the cells were centrifuged at 800rcf for 5 min, the medium was aspirated, and the cells were resuspended in a phosphate-buffered saline (PBS) solution. This was then mixed in a 1:1 ratio with Matrigel (Corning and BD Biosciences) and transplanted to each mouse using a 100 μL injection subcutaneously. The tumor was monitored for growth up until the size of 12–15 mm, which is the size at which a treatment would begin. The mice were continuously monitored for tumor size throughout the course of the treatment and euthanized if the tumor size ever reached the 20 mm limit, as recommended by the Administrative Panel on Laboratory Animal Care (APLAC) guidelines.

The Tubulin Polymerization Assay (EMD Millipore) was used to confer the mechanism of azapodophyllotoxins, following the protocol for the Tubulin Polymerization Assay kit (Sigma 17-10194). The details can be found in the Supporting Information.

NIA was performed using the ProteinSimple Nanopro 1000 machine. The final protein concentration loaded into each nanofluidic capillary of the machine was 0.1 μg/μL. The primary antibody for tubulin (Cell Signaling Technologies) was diluted 1:100. The primary antibody for HSP70 (Santa Cruz Biosciences) was diluted 1:500. The secondary antirabbit and antimouse HRP-conjugates were both diluted 1:100. A chemiluminescence signal was recorded after the addition of the detection reagent, which consists of a 1:1 ratio of luminol and peroxide. An analysis of the chemiluminescence data from NIA was performed via the ProteinSimple Compass software. FNA and the core biopsy protocol can be found in supplement.

DESI-MSI functions by directing an electrospray of pneumatically assisted solvent microdroplets onto the surface of interest.12 The electrosprayed solvent microdroplets dissolve certain chemical constituents off the surface investigated and also induce the formation of secondary—charged—droplets that take off from the surface. These secondary droplets already contain constituents of the sample and produce molecular ions of the analytes upon evaporation in the heated inlet of the mass spectrometer. In this way, electrospray-like mass spectrometric information is obtained on the sample, featuring multiply charged ions and solvent adducts among other electrospray-specific spectral features.

Acknowledgments

We thank current and former members of the Felsher, Malhotra, and Zare laboratories for their helpful suggestions during this project. We thank B. Rosellini and R. Barros for their help. We are very grateful for the SPARK program advisors, especially Dr. S. Schow and Dr. R. Greenhouse. A.M.G. and D.W.F. are grateful for the support of Stanford’s SPARK Translational Research Program, Stanford Clinical and Translational Science Award (CTSA) program and Spectrum (UL1TR003142). A.M.G. and K.M. are grateful to the Stanford Cancer Translational Nanotechnology Training (TNT) T32 training grant funded by the National Cancer Institute (T32 CA196585) and the Stanford Center of Molecular Analysis and Design, respectively. A.R. is thankful to the Stanford Clinical and Translational Science Award (CTSA) for T32 training grant funded by the National Center for Advancing Translational Sciences (NCATS). This work is supported by the National Institutes of Health under R01 CA184384 (D.W.F. and R.N.Z.), U01 CA188383 (D.W.F.), R01 CA208735 PQ7 (D.W.F.), R35CA253180 (D.W.F.), and R01 DK114174 (S.V.M.).

Glossary

Abbreviations

AZP

Azapodophyllotoxins

DESI-MS

Desorption Electrospray Ionization Mass Spectrometry

DESI-MSI

Desorption Electrospray Ionization Mass Spectrometry Imaging

FNA

Fine Needle Aspirates

MCR

Multicomponent Reaction

MS

Mass Spectrometry

NIA

Nanoimmunoassay/nanofluidic proteomic immunoassay

NSG

NOD-scid gamma mice

RCC

Renal Cell Cancer

SAR

Structure Activity Relationship

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00673.

  • Synthesis methods for AZP derivatives, in vitro and in vivo kidney cancer data, and quantification of tubulin by nanoimmunoassay (PDF)

Author Contributions

A.M.G., D.W.F., S.V., and R.N.Z. conceived and designed the experiments. A.M.G., V.K., A.R., F.B.A., N.S.L., and K.M. performed the experiments. A.M.G., N.S.L., F.B.A., K.M., and L.T. analyzed the data. D.W.F., S.V., and R.N.Z. contributed reagents, materials, and analysis tools. A.M.G., F.B.A., V.K., K.M., L.T., S.V., and D.W.F. wrote the paper.

The authors declare no competing financial interest.

Supplementary Material

ml1c00673_si_001.pdf (253.5KB, pdf)

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