Tightly regulated activity of the transcription factor MYC is essential for orderly cell proliferation. Upon deregulation, MYC elicits and promotes cancer progression. Proteasomal degradation is an essential element of MYC regulation, initiated by phosphorylation at Serine62 (Ser62) of the MB1 region. Here, we found that Ser62 phosphorylation peaks in mitosis, but that a fraction of nonphosphorylated MYC binds to the microtubules of the mitotic spindle. Consequently, the microtubule-destabilizing drug vincristine decreases wild-type MYC stability, whereas phosphorylation-deficient MYC is more stable, contributing to vincristine resistance and induction of polyploidy. PI3K inhibition attenuates postmitotic MYC formation and augments the cytotoxic effect of vincristine.

Implications:

The spindle's function as a docking site for MYC during mitosis may constitute a window of specific vulnerability to be exploited for cancer treatment.

The nuclear bHLH-Zip transcription factor MYC regulates gene expression programs that govern fundamental cellular processes including proliferation (1–4). The control of MYC activity is tight, with an estimated half-life of MYC protein in the range of 20 to 30 minutes (5–9). MYC protein stability is critically regulated by phosphorylation events in the MB1 box region of the N-terminal transcriptional regulatory domain (10–12). Phosphorylation at Serine62 (Ser62) has been shown to stabilize MYC protein and transcriptional activity (13), whereas subsequent phosphorylation at Threonine58 (Thr58) initiates ubiquitin-mediated proteasomal degradation (11, 14, 15). MYC phosphorylation has been described to occur in mitosis, and several kinases have been involved including the cyclin-dependent kinases Cdk1 and Cdk2 (8, 16, 17). Thus in neuronal precursor cells, Ser54, the paralog MB1 serine phosphorylation site of N-MYC, is phosphorylated by Cdk1 in mitosis-initiating subsequent growth arrest and differentiation of the daughter cells (18). At the same time, MYC activity is a critical determinant of mitotic cell fate, shown to modulate the survival of tumor cells exposed to chemotherapy and to regulate cell fate following asymmetric cell division of antigen-stimulated T cells (19, 20). Given its pivotal role for mitotic progression and proliferation, we here addressed the fate of MYC protein during mitosis in cancer cells and asked for interactions with cytotoxic drugs predominantly acting in mitosis.

Cells, tumor model, and small molecules

Lymphoma and cancer cell lines were obtained from public depositories or cooperating groups as listed (Supplementary Materials and Methods). Short-tandem repeat–profiled cells were cultured to obtain frozen master stocks, from which cells were thawed and expanded for experimentation. All cell lines were cultured at 37°C with 5% CO2 in RPMI1640 or DMEM medium containing 10% or 20% FCS (Gibco-BRL) and 1% penicillin/streptomycin (Sigma, Biochrom). Cell cultures were regularly tested for mycoplasma contamination with a PCR-based universal mycoplasma detection kit (VenorGeM, Minerva) according to kit instructions, and cells from mycoplasma-free frozen master stocks negative cultures were used for experimentation. Animal experiments were conducted in accordance with Institutional Animal Care and Use Committee–approved protocols at Beth Israel Deaconess Medical Center. TA lymphoma experiments were performed using a well-established mouse model of cancer, in which tumors arising spontaneously in K14-Cre+ Brca1f/f;Trp53f/f mice are orthotopically transplanted into K14-Cre–negative genetically identical littermates, as previously described (21). Dimethylanthrone (DME) and cycloheximide (CHX) were obtained from Sigma-Aldrich, and pharmaceutical grade vincristine and paclitaxel were purchased from Cell-Pharm and Hexal, respectively. The PI3K inhibitors NVP-BKM120 (BKM120), idelalisib (CAL-101), and copanlisib were obtained or purchased from Selleckchem, Gilead Sciences, and Bayer AG, respectively. Further information on antibodies, small molecules, plasmids, and vectors is provided in the Supplementary Data section. Flow cytometry, cell sorting, confocal microscopy, SDS-PAGE, immunoprecipitation, Western blot, viability tests, RNA preparation, PCR, and colony formation assays in semi-solid methylcellulose were performed according to standard protocols (Supplementary Materials and Methods).

Nuclear magnetic resonance spectroscopy

All nuclear magnetic resonance (NMR) experiments were acquired using a 900 MHz spectrometer equipped with a 5-mm triple resonance cryoprobe. The transfer Nuclear Overhauser enhancement (trNOE) experiments were done using spectral windows of approximately 14 ppm in t1 and t2 dimensions with a mixing time of 350 ms, 64 scans per transient, and 400 t1 increments per experiment. Saturation transfer difference (STD) experiments were done using a standard Bruker pulse sequence, acquiring 16k data points with a saturation time of 2 seconds. The spectra were processed and analyzed using TopSpin (Bruker BioSpin).

Statistical evaluations

Statistical tests were performed using Excel 2013 or GraphPad Prism version 4.03 for Windows as indicated (Microsoft Excel 2013, GraphPad Software, www.graphpad.com), and P values are given for each experiment. Error bars represent SDs of mean values.

MYC is phosphorylated at Ser62 in mitosis, but a fraction of nonphosphorylated MYC binds to tubulin microtubules of the mitotic spindle apparatus

As MYC degradation requires prior phosphorylation at Ser62 and then Thr58 of the MYC-Box I (MBI) region (15), we measured total and phosphorylated MYC using flow cytometry. We found that total MYC was evenly detectable throughout the cell cycle (Fig. 1A, top plot). Phospho-specific MYC antibodies revealed increased phosphorylation at Ser62 (pS62) and Thr58 (pS62/pT58) distinctly in G2–M cells with high total MYC content, with larger populations of pS62-positive compared with pS62/pT58-dual–positive cells (Fig. 1A, as boxed, Fig 1B; Supplementary Fig. S1A). Ser62 phosphorylation of MYC was associated with phosphorylation of the mitosis marker MPM2 in all cell types examined, including the diffuse large B-cell lymphoma cell line OCI-Ly3 (Fig. 1A), a panel of leukemia, lymphoma, and solid cancer cell lines, as well as in proliferating nontransformed cells such as murine embryonic fibroblasts, CD34-positive human hematopoietic stem cells, and mitogen-stimulated human T cells (Supplementary Fig. S1A). Consistent with these cell line observations, we detected a tight correlation of pSer62-MYC with Ser10 phosphorylation in histone H3, also phosphorylated predominantly during mitosis, in clinical specimen of patients with primary aggressive lymphoma (Supplementary Fig. S1C and S1D). At the subcellular level, we found that pSer62-MYC did not localize to chromosomal DNA in metaphase cells, but rather spread evenly throughout the cytoplasm, from where it could be released by cell membrane perforation with digitonin (Supplementary Fig. S1E).

Figure 1.

MYC is phosphorylated at Ser62 and Thr58 in mitosis, and nonphosphorylated MYC binds to the mitotic spindle. A, Flow cytometry of MYC versus DNA content during the cell cycle in OCI-Ly3 lymphoma cells, cells with strong MYC phosphorylation at Ser62 and S62er/Thr58 are boxed. Middle plot: pSer62-MYC and pThr58-MYC versus total MYC. Bottom plot: mitosis marker pMPM2 versus pSer62-MYC and pSer62/pThr58-MYC, double-positive cells as boxed, isotype controls inserted. B, Quantification of pSer62-MYC–positive and pThr58-MYC–positive cells from OCI-Ly3 (n = 3) and U2932 (n = 7), paired two-sided t test with Bonferroni correction. C, Immunoblots for MYC and pSer62-MYC from HeLa and Hek293 cells, asynchronous or arrested in mitosis with DME. D, Immunoblots for pSer62-MYC with and without AP treatment with and without AP inhibition by sodium orthovanadate (Na3VO4) from mitotic Hek293 cells. E, Immunoblots of MYC and pSer62-MYC from equal number of asynchronous or mitotic HeLa or HCT116 cells after thymidine-synchronization, release, and shake-off at the indicated time points after mitosis (M = 0 hours). F, Immunoblots of MYC and pSer62-MYC during and after mitosis with and without addition of CHX. G, Confocal microscopy of HeLa cells stained against MYC (MoAb Y69) and pMPM2; scale bar, 10 μm; representative example of n = 3. H, Coprecipitation of MYC with tubulin microtubules from mitotic cells separated by sequential centrifugation, schematic overview in the top plot. I, Coimmunoprecipitation of MYC and p-Ser62-MYC with TUBA in lysates from HeLa cells and coimmunoprecipitation of tubulin with MYC, asynchronous or synchronized by exposure to DME for 16 hours (I, input; P, precipitate, rabbit polyclonal N262 used for recognition of MYC, rabbit MoAb E1J4K for pSer62-MYC, murine MoAb B-5-1–2 for TUBA; murine and rabbit immunoglobulin (mIg and rIg) as controls).

Figure 1.

MYC is phosphorylated at Ser62 and Thr58 in mitosis, and nonphosphorylated MYC binds to the mitotic spindle. A, Flow cytometry of MYC versus DNA content during the cell cycle in OCI-Ly3 lymphoma cells, cells with strong MYC phosphorylation at Ser62 and S62er/Thr58 are boxed. Middle plot: pSer62-MYC and pThr58-MYC versus total MYC. Bottom plot: mitosis marker pMPM2 versus pSer62-MYC and pSer62/pThr58-MYC, double-positive cells as boxed, isotype controls inserted. B, Quantification of pSer62-MYC–positive and pThr58-MYC–positive cells from OCI-Ly3 (n = 3) and U2932 (n = 7), paired two-sided t test with Bonferroni correction. C, Immunoblots for MYC and pSer62-MYC from HeLa and Hek293 cells, asynchronous or arrested in mitosis with DME. D, Immunoblots for pSer62-MYC with and without AP treatment with and without AP inhibition by sodium orthovanadate (Na3VO4) from mitotic Hek293 cells. E, Immunoblots of MYC and pSer62-MYC from equal number of asynchronous or mitotic HeLa or HCT116 cells after thymidine-synchronization, release, and shake-off at the indicated time points after mitosis (M = 0 hours). F, Immunoblots of MYC and pSer62-MYC during and after mitosis with and without addition of CHX. G, Confocal microscopy of HeLa cells stained against MYC (MoAb Y69) and pMPM2; scale bar, 10 μm; representative example of n = 3. H, Coprecipitation of MYC with tubulin microtubules from mitotic cells separated by sequential centrifugation, schematic overview in the top plot. I, Coimmunoprecipitation of MYC and p-Ser62-MYC with TUBA in lysates from HeLa cells and coimmunoprecipitation of tubulin with MYC, asynchronous or synchronized by exposure to DME for 16 hours (I, input; P, precipitate, rabbit polyclonal N262 used for recognition of MYC, rabbit MoAb E1J4K for pSer62-MYC, murine MoAb B-5-1–2 for TUBA; murine and rabbit immunoglobulin (mIg and rIg) as controls).

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Upon gel electrophoresis, MYC phosphorylated during mitosis migrated at slightly slower speed, and was separated in a major and a minor band (Fig. 1C), presumably due to conformational changes associated with phosphorylation. Similar observations had been made for the corresponding Ser54 phosphorylation site in the MB1 box of N-MYC (18). As controls for the specificity of the anti-MYC and the anti–pSer62-MYC antibodies, we found no binding of the anti-MYC antibody Y69 nor the anti-pSer62 antibody to c-myc−/− HO15.19 cells (Supplementary Fig. S2A), as well as no binding of the phospho-specific antibody to phosphorylation-deficient c-MYC-S62A even at high expression levels (Supplementary Fig. S2B). Immunoblot detection of pSer62-MYC in mitotic cells was sensitive to alkaline phosphatase (AP) treatment, both from endogenous and ectopic MYC expression in Hek293 cells (Fig. 1D; Supplementary Fig. S2C). We then analyzed the levels of MYC and pSer62-MYC in synchronized cells. Following enrichment in mitosis by thymidine synchronization, 8-hour release, and harvest of nonadherent cells, we noted a decrease of MYC at 2 hours after mitosis, which was replenished by CHX-sensitive de novo protein biosynthesis (Fig. 1E and F). We then applied the mitotic kinesin Eg5/Kif11 inhibitor DME to accumulate cells in mitosis and found postmitosis MYC levels decreased to approximately one third at 2 hours after release from mitotic arrest, as quantified by Western blot in HeLa and HCT116 cells (MoAb Y69, HeLa: 30.6% ± 16.4%, HCT116: 41.0% ± 28.1%, n = 3 each). Importantly, DME arrests cells by inhibiting spindle pole segregation, but leaves the microtubule architecture intact (22). This feature separates DME from most G2–M arresting drugs such as nocodazole, colcemid-derivatives, and vinca-alkaloids, which destroy the spindle. Hence, we used DME as a means to achieve “microtubule-sparing” G2–M arrest.

We then stained cells under conditions that preserved microtubule integrity. We discovered that MYC associated with the mitotic spindle apparatus, evident from metaphase through telophase (spontaneous mitotic HeLa cell in Figs. 1G, 2E, and 3A and B). The stains were most clearly defined when using the monoclonal rabbit antibody Y69, a high avidity antibody directed against an epitope within the first 100 amino acids of MYC. Similar staining was seen using a polyclonal rabbit antibody directed against N-terminal (ab N262) and a murine monoclonal antibody against C-terminal (ab 9E10) epitopes (Supplementary Fig. S3A). In addition, we confirmed the finding of MYC association with the spindle by electron microscopy (Supplementary Fig. S3C). MYC had previously been shown to interact with tubulin outside mitosis (23, 24). Applying the method of Alexandrova and colleagues (23) to demonstrate MYC microtubule interactions in vivo in mitosis, we fractionated lysates of mitotic HeLa cells by differential centrifugation which separates MYC bound to polymerized tubulin (P2) from MYC bound to nuclear debris (P1) and soluble MYC in the supernatant (SN2; Fig. 1H). The majority of intact MYC precipitated at 25,000 × g in the P2 fraction, containing the tubulin polymers of the spindle, while in contrast, pSer62-MYC was found predominantly in the supernatant (Fig. 1H; ref. 25). These results clearly confirm the binding of MYC to tubulin microtubules in vivo (23, 24), but importantly, they extend this interaction also to the situation in mitosis. Consistently, immunoprecipitation of tubulin alpha (TUBA) coimmunoprecipitated MYC in lysates of asynchronous and DME-arrested cells, and MYC pull-down coimmunoprecipitated TUBA. However, an immunoprecipitation of TUBA did not precipitate pSer62-MYC from asynchronous cells, and only at low levels from mitotic cells (Fig. 1I).

Figure 2.

MYC phosphorylation at Serin62 and Threonin58 modulates binding to tubulin. A, Design of MYC-MBI peptide comprising aa 56–68, with and without PO3H2 modification of Ser62 or Thr58, respectively. B, Binding of MYC-MBI peptides to tubulin as probed by NMR. Trace corresponding to the L56/61Hδ1,δ2 to L56/61Hγ and L56/61Hβ of a representative trNOE experiment. C, Signal volume of bound peptide population, depending on PO3H2 modification at either Thr58 (pT58, green) or Ser62 (pS62, red), compared with the unmodified control (WT, blue). D, Binding prediction of MYC-MBI (56–68) to the tubulin alpha–beta dimer. Superimposed ensemble of the 10 best fitting predictions of MYC-MBI (56–68) to tubulin (RPBS). E, Immunofluorescence stains of HCT116 cells after 2 hours release from DME-induced G2–M arrest with and without CHX, nuclear DAPI counterstain as inserted. Scale bar, 50 μm. F, Flow cytometry of HCT116 cells treated as above, detecting MYC with and without CHX in the cell population in G0–G1 at 2 hours after release from DME, as boxed. G, Immunoblots of Hek293 cells transfected with plasmids expressing c-myc wt or the variants c-myc-Ser62A, -Thr58A. Twenty-four hours after transfection, the cells were propagated for 16 hours without DME (asynchronous) or with DME (mitotic), followed by release from arrest in the presence of CHX for MYC and pSer62-MYC, representative example of triplicates. H, Coprecipitation of MYC with tubulin microtubules from Hek293 cells, 48 hours after transfection with c-myc wt or the variants, with DME treatment as indicated. Mean values of three replicates, two-way ANOVA with Tukey multiple comparisons test. I, Immunoblot of MYC with and without DME-mediated mitotic arrest in HeLa cells with stable MYC-S62A or MYC-T58A expression. Quantification of MYC before or after release from DME in the presence of CHX as indicated. Mean values of three replicates, one-way ANOVA with Dunnett's multiple comparisons test.

Figure 2.

MYC phosphorylation at Serin62 and Threonin58 modulates binding to tubulin. A, Design of MYC-MBI peptide comprising aa 56–68, with and without PO3H2 modification of Ser62 or Thr58, respectively. B, Binding of MYC-MBI peptides to tubulin as probed by NMR. Trace corresponding to the L56/61Hδ1,δ2 to L56/61Hγ and L56/61Hβ of a representative trNOE experiment. C, Signal volume of bound peptide population, depending on PO3H2 modification at either Thr58 (pT58, green) or Ser62 (pS62, red), compared with the unmodified control (WT, blue). D, Binding prediction of MYC-MBI (56–68) to the tubulin alpha–beta dimer. Superimposed ensemble of the 10 best fitting predictions of MYC-MBI (56–68) to tubulin (RPBS). E, Immunofluorescence stains of HCT116 cells after 2 hours release from DME-induced G2–M arrest with and without CHX, nuclear DAPI counterstain as inserted. Scale bar, 50 μm. F, Flow cytometry of HCT116 cells treated as above, detecting MYC with and without CHX in the cell population in G0–G1 at 2 hours after release from DME, as boxed. G, Immunoblots of Hek293 cells transfected with plasmids expressing c-myc wt or the variants c-myc-Ser62A, -Thr58A. Twenty-four hours after transfection, the cells were propagated for 16 hours without DME (asynchronous) or with DME (mitotic), followed by release from arrest in the presence of CHX for MYC and pSer62-MYC, representative example of triplicates. H, Coprecipitation of MYC with tubulin microtubules from Hek293 cells, 48 hours after transfection with c-myc wt or the variants, with DME treatment as indicated. Mean values of three replicates, two-way ANOVA with Tukey multiple comparisons test. I, Immunoblot of MYC with and without DME-mediated mitotic arrest in HeLa cells with stable MYC-S62A or MYC-T58A expression. Quantification of MYC before or after release from DME in the presence of CHX as indicated. Mean values of three replicates, one-way ANOVA with Dunnett's multiple comparisons test.

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Figure 3.

Tubulin-interacting drugs paclitaxel or vincristine modulate postmitotic protein levels. A, Immunoblots for MYC from HeLa and BL-41 cells, asynchronous or arrested in G2–M by DME and exposed to increasing concentrations of vincristine or paclitaxel as indicated and MYC levels, mean values of three replicates, one-way ANOVA with Dunnett's multiple comparisons test. B, Immunofluorescence of TUBA and MYC in HeLa cells arrested in G2–M by DME for 20 hours, in parallel treated with 3 μmol/L vincristine or paclitaxel, representative example of three replicates; scale bar, 10 μm. C, MYC coprecipitation with microtubules (P2) in HeLa cells, accumulated in mitosis by DME and coincubation with 20 nmol/L vincristine or paclitaxel. D, MYC abundance in the P2 precipitate, normalized to total GAPDH (P2+SN2), mean values of three replicates, two-way ANOVA with Tukey multiple comparison test.

Figure 3.

Tubulin-interacting drugs paclitaxel or vincristine modulate postmitotic protein levels. A, Immunoblots for MYC from HeLa and BL-41 cells, asynchronous or arrested in G2–M by DME and exposed to increasing concentrations of vincristine or paclitaxel as indicated and MYC levels, mean values of three replicates, one-way ANOVA with Dunnett's multiple comparisons test. B, Immunofluorescence of TUBA and MYC in HeLa cells arrested in G2–M by DME for 20 hours, in parallel treated with 3 μmol/L vincristine or paclitaxel, representative example of three replicates; scale bar, 10 μm. C, MYC coprecipitation with microtubules (P2) in HeLa cells, accumulated in mitosis by DME and coincubation with 20 nmol/L vincristine or paclitaxel. D, MYC abundance in the P2 precipitate, normalized to total GAPDH (P2+SN2), mean values of three replicates, two-way ANOVA with Tukey multiple comparison test.

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In summary, we found that in mitosis a proportion of MYC, detached from DNA, is phosphorylated at Ser62 and subsequently at Thr58. A fraction of nonphosphorylated MYC protein, however, binds to the tubulin of the mitotic microtubules, i.e., the spindle.

MYC phosphorylation at Ser62 and Thr58 modulates binding to tubulin

To analyze protein interactions of tubulin and the MYC MB1 region directly, we designed oligopeptides corresponding to amino acid 56 through 68 of MYC [MYC-MBI (56–68)], with and without Ser62 and Thr58 phosphorylation (Fig. 2A). NMR spectroscopy, specifically trNOE experiments, revealed that the unmodified peptide bound to tubulin alpha–beta heterodimers. Binding decreased when modifications were introduced that mimic phosphorylation at Ser62 or Thr58, respectively, by at least a factor of 10 (Fig. 2B and C). We then titrated the peptide competitively against epothilone-A, a ligand to tubulin dimers with a known dissociation constant of 150 μmol/L (26). In these experiments, abolishment upon binding can be used to determine binding affinities (Kd), given a ligand of known affinity is available for the same system. In this case, we have studied epothilone-A bound to tubulin allowing us to determine the Kd of MYC-MBI (56–68) to microtubules to be between 77 ± 7 μmol/L and 565 μmol/L (see Supplementary Information for details). These estimates are in the range of previously reported values for the binding of Tau peptides to microtubules (27) or the binding of MYC-MBI–related peptides to other proteins such as bridging integrator protein 1 (BIN1; ref. 28) or peptides of N-MYC to the kinase Aurora-A (29).

We then mapped the MYC-MBI (56–68) peptide sequence using the Resource Parisienne en Bioinformatique Structurale (RPBS) data bank to obtain binding predictions to the tubulin alpha-/beta tubulin dimer (RPBS, Alland, 2005; refs. 30, 31). Eight of the ten best fitting predictions clustered at the plus-end site of the tubulin alpha–beta heterodimer (Fig. 2D), consistent with binding of the MYC-MB1 region to tubulin close to the dimer interface.

The protection of spindle-bound MYC protein from degradation raises the possibility that it is carried over into the newly formed cells. To examine this possibility, we released cells from mitotic arrest, and measured MYC in sorted postmitotic cells. At 2 hours after mitosis, we found MYC clearly detectable in the nuclei by confocal microscopy, and significant staining remained after suppression de novo protein synthesis in the newly formed cells (Fig. 2E and F), suggesting that a fraction of MYC had been derived from the parental cells. CHX, which we have employed to inhibit protein biosynthesis, may interfere with the stability of proteins. Although we have not seen MYC stabilization in the corresponding immunoblots (Figs. 1F and 2G), we cannot exclude that CHX may have contributed to MYC stability. We then followed MYC levels from ectopic c-myc-wt or the phosphorylation-deficient variants in asynchronous and mitotic Hek293 cells. In the presence of CHX, MYC levels progressively declined in asynchronous cells and in cells released from mitotic arrest, but at a slower pace in the S62A and T58A variants (Fig. 2G). The analysis of the P2 pellet after cell fragmentation then showed that enforced mitotic arrest led to a significant loss of ectopic wt MYC in complex with tubulin polymers, but not for the Ser62- and Thr58-MYC variants (Fig. 2H). As a prerequisite for functional studies without effects of endogenous MYC, we then created cell lines expressing phosphorylation-deficient MYC-T58A and -S62A after extinguishing endogenous MYC expression. Effective silencing of endogenous MYC was achieved with a 3-UTR-MYC-sh-vector (Supplementary Fig. S4C). MYC depletion killed most cells within 7 days and abrogated cell growth completely in HeLa, Hek293, and P493-6 cells (Supplementary Fig. S4D). However, we rescued HeLa cells by expression of MYC-S62A or MYC-T58A, and then we accumulated HeLa-sh-3UTR-MYC/+pMYC-S62A (HeLa-S62A; Supplementary Fig. S4E), HeLa-sh-3UTR-MYC/+pMYC-S58A (HeLa-S58A), and HeLa control cells in mitosis, and followed MYC protein levels during release in the presence of CHX. MYC levels were higher in the variant cell lines and dropped in all cell types, albeit with persistence of higher MYC protein levels in the phosphorylation-deficient MYC-S62A or MYC-T58A variants (Fig. 2I). These data are compatible with slowed MYC decay by phosphorylation-deficient Ser62 and Thr58 mutations both in asynchronous and in mitotic cells.

Taken together, we propose that MYC's binding to tubulin in the mitotic spindle apparatus is modulated by phosphorylation-sensitive interactions at the MB1 (56–68) of MYC.

Tubulin-interacting vincristine destabilizes mitotic MYC

The observation of MYC transfer through mitosis via the spindle raised our interest in the functional consequences of this mechanism, in particular for cell division in cancer. Substances affecting spindle microtubule dynamics are among the most efficient cytotoxic drugs known (32, 33). They induce death in mitosis, but differ in their mechanism of action: vincristine destabilizes microtubules, and paclitaxel stabilizes microtubules (34). We hypothesized that drugs that interfere with microtubule stability should also affect the stability of MYC, because they would modulate the integrity of the tubulin scaffold. Vincristine would be expected to destabilize MYC in mitosis toward increased decay, whereas paclitaxel would not. This effect should differ between cell populations with low mitotic indices from asynchronous cell cultures, and cell populations with high mitotic index, e.g., after DME arrest. Noteworthy, vincristine and paclitaxel alone already arrest cells in mitosis (35), but we expected that preaccumulation of the cells in G2–M by DME pretreatment should greatly enhance the drug effects in mitotic cells. We found that treatment with paclitaxel in fact did not change MYC protein levels in asynchronous and DME-treated HeLa cells, whereas MYC levels in Burkitt lymphoma cells were slightly reduced in DME-treated cells (Fig. 3A). In contrast, vincristine clearly diminished MYC in cell populations arrested in mitosis after DME treatment (“mitotic”) versus untreated populations (“asynchronous”; Fig. 3A; Supplementary Fig. S5A). Confocal microscopy imaging confirmed that cells treated with paclitaxel after DME-mediated G2–M arrest display typical monaster-shaped spindles with positive staining for MYC, whereas vincristine destabilized both spindle microtubules and MYC (Fig. 3B). For further proof, in situ microtubule precipitation experiments showed that vincristine abrogated the coprecipitation of MYC with tubulin, which was not altered by paclitaxel (Fig. 3C and D). Noteworthy, vincristine did not prevent tubulin pellet formation completely (Fig 3C), suggesting residual microtubule formation or interference from other proteins of the cellular extract. We then addressed the impact of vincristine exposure during mitosis on gene transcription. As early as 2 hours after mitosis, there was no immediate effect of vincristine: the expression levels of 89 MYC target genes as well as of MYC itself were not altered in sorted viable postmitotic cells, except reduced levels of branched chain amino acid transaminase 1 transcripts in HCT116 cells (Supplementary Fig. S4B and S4C).

Vincristine and paclitaxel differ in postmitotic cytotoxicity

We next compared the features of cell killing by paclitaxel and vincristine during and after mitosis in a panel of B-cell lymphoma cell lines. We found that both vincristine and paclitaxel induced death of cells in G2–M in a dose-dependent manner, with higher efficacy of vincristine, particularly at low concentrations (Fig. 4A and B). Still, we observed a fraction of viable cells to persist after 72-hour exposure to both drugs (boxed in gray, Fig. 4A, bottom plot). To analyze the fate of these persistent cells, we employed colony formation assays as a measure of single-cell regeneration capacity. In these experiments, the effects of vincristine and paclitaxel diverged dramatically. After exposure to vincristine for 72 hours, colony numbers were significantly lower than after exposure to paclitaxel (Fig. 4C and D). When we calculated the effect ratios of vincristine versus paclitaxel at equimolar concentrations, we found a significantly stronger effect of vincristine on colony forming unit (CFU) inhibition (Fig. 4E). The strong suppressive effects of vincristine on lymphoma colony formation were evident in cells with both high and low MYC expression. But, as shown in the P493-6 system, colony suppression by vincristine was much more pronounced when cells expressed high levels of MYC (Fig. 4F). These experiments showed that vincristine and paclitaxel both induced direct death in G2–M with comparable efficacy. In addition, and clearly distinct, vincristine exerted a strong postdrug exposure inhibitory effect on clonal regeneration in the CFU assays. Noteworthy, this effect was not be detected by standard short-term viability tests. However, this effect should be pertinent to the patient situation, where relapse or resistant disease originates from regenerating tumor cells.

Figure 4.

Vincristine kills lymphoma cells in G2–M more effectively than paclitaxel and inhibits postcytostatic clonogenic regrowth. A, Cell death and cell-cycle distribution in P493-6 cells directly following drug exposure to paclitaxel or vincristine for 72 hours. Propidium iodide (PI)-positive dead cells represented in red, viable cells in gray, percentages of dead cells inserted in red, and untreated control inserted in top right plot. B, Cell death by flow cytometry from 13 BL and 3 DLBCL cell lines exposed to 2, 10, and 20 nmol/L paclitaxel or vincristine for 72 hours. Cell death by flow cytometry, mean values of triplicates of all cell lines, two-way ANOVA. C, Colony density and morphology of OCI-Ly3 lymphoma cells at day 10 of culture in semisolid media, following exposure of 2.5 × 104 cells to 2 nmol/L paclitaxel or 2 nmol/L vincristine for 72 hours; control: 2.5 × 102 untreated cells; scale bar, 100 μm. D, Colony numbers at day 10 in semisolid media from 9 Burkitt lymphoma (BL) and 3 DLBCL lymphoma cell lines exposed to vincristine or paclitaxel for 72 hours, washed and seeded in semisolid methylcellulose. Mean ± SD of independent triplicates, unpaired two-sided t test, *, P < 0.05; § = no growth. E, Ratios of effects for direct cytotoxicity (DCT) and colony formation (CF) exerted by vincristine versus paclitaxel. For DCT, the ratio of mean percentages of dead cells after vincristine and paclitaxel; for CF, the number of colonies obtained at day 10. F, Colony numbers at day 10 from MYC-low and MYC-high P493-6 lymphoma cells. In MYC-low cells, MYC was reduced by 24-hour preincubation with doxycycline. Cells (25 × 103) were exposed to vincristine or paclitaxel for 72 hours, washed, and seeded in semisolid methylcellulose without doxycycline. Mean ± SD of triplicates, representative example of three independent experiments paired two-sided t tests with Bonferroni correction.

Figure 4.

Vincristine kills lymphoma cells in G2–M more effectively than paclitaxel and inhibits postcytostatic clonogenic regrowth. A, Cell death and cell-cycle distribution in P493-6 cells directly following drug exposure to paclitaxel or vincristine for 72 hours. Propidium iodide (PI)-positive dead cells represented in red, viable cells in gray, percentages of dead cells inserted in red, and untreated control inserted in top right plot. B, Cell death by flow cytometry from 13 BL and 3 DLBCL cell lines exposed to 2, 10, and 20 nmol/L paclitaxel or vincristine for 72 hours. Cell death by flow cytometry, mean values of triplicates of all cell lines, two-way ANOVA. C, Colony density and morphology of OCI-Ly3 lymphoma cells at day 10 of culture in semisolid media, following exposure of 2.5 × 104 cells to 2 nmol/L paclitaxel or 2 nmol/L vincristine for 72 hours; control: 2.5 × 102 untreated cells; scale bar, 100 μm. D, Colony numbers at day 10 in semisolid media from 9 Burkitt lymphoma (BL) and 3 DLBCL lymphoma cell lines exposed to vincristine or paclitaxel for 72 hours, washed and seeded in semisolid methylcellulose. Mean ± SD of independent triplicates, unpaired two-sided t test, *, P < 0.05; § = no growth. E, Ratios of effects for direct cytotoxicity (DCT) and colony formation (CF) exerted by vincristine versus paclitaxel. For DCT, the ratio of mean percentages of dead cells after vincristine and paclitaxel; for CF, the number of colonies obtained at day 10. F, Colony numbers at day 10 from MYC-low and MYC-high P493-6 lymphoma cells. In MYC-low cells, MYC was reduced by 24-hour preincubation with doxycycline. Cells (25 × 103) were exposed to vincristine or paclitaxel for 72 hours, washed, and seeded in semisolid methylcellulose without doxycycline. Mean ± SD of triplicates, representative example of three independent experiments paired two-sided t tests with Bonferroni correction.

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We next addressed the contribution of MYC phosphorylation at Ser62 and Thr58 to the specific cytotoxic effect exerted by vincristine. We exposed HeLa-S62A and HeLa-T58A cells, as described above, to vincristine during mitosis. Although in the parental cells vincristine induced significant MYC decline as expected, the variants with phosphorylation-deficient MYC mutations retained MYC protein levels in the presence of vincristine following accumulation in mitosis (Fig. 5A). In addition, persistence of phosphorylation-deficient MYC was associated with improved viability after 72-hour exposure to vincristine (Fig. 5B), albeit with significant morphologic alterations in the drug-exposed cells. Although the parenteral HeLa cells died with increasing vincristine concentrations, a fraction of the cells with phosphorylation-deficient MYC survived and formed clusters of enlarged cells (Fig. 5C and D). Analyzing the cytogenetic status of these cells, we then found that in the surviving cells with phosphorylation-deficient MYC, the number of chromosomes had doubled after 72 hours of exposure to ≥20 nmol/L vincristine (Fig. 5E and F). The occurrence of tetraploid metaphases clearly documented that the MYC-S62A and MYC-T58A cells had overcome mitotic arrest without actually dividing, had undergone one round of cell cycling, and had re-entered mitosis during the 72-hour exposure to vincristine. We conclude that stabilization of MYC through introduction of the S62A and T58A mutations allowed cells to survive microtubule disruption caused by vincristine during mitosis, and to enter into a subsequent cell cycle with a double set of chromosomes, leading to polyploidy of these cancer cells.

Figure 5.

S62A and T58A mutations impede vincristine-associated mitotic MYC degradation and mediate resistance to vincristine. A, Immunoblot of MYC in HeLa cells, after silencing endogenous MYC and expression of MYC-S62A and MYC-T58A. Accumulation in mitosis by DME for 16 hours, with or without vincristine in parallel. Mean values of three independent experiments, two-sided t test with Bonferroni correction. B, Viability of HeLa cells measured by MTS exposed to vincristine for 72 hours at the concentration indicated, mean values from three independent experiments, each run in triplicates. Two-sided t test, P value < 0.05 (***) according to Holm–Sidak method for HeLa-wt versus HeLa-S62A and HeLa-T58A for vincristine concentrations ≥ 10 nmol/L. C, Morphology of HeLa-MYC-S62A and HeLa-T58A and parental HeLa-wt cells incubated with vincristine with 10 and 20 nmol/L vincristine for 72 hours, washed and seeded in semisolid medium. Hela-S62A and HeLa-T58A cells formed colonies at day 7, whereas HeLa-wt cells showed few large, atypical cells after 10 nmol/L vincristine, and no viable cells after 20 nmol/L vincristine; scale bar, 100 μm. D, Cell viability by PI- and Hoechst33342 directly after 72-hour vincristine incubation. E, Hyperdiploid metaphases of HeLa-MYC-S62A and HeLa-MYC-T58A cells after 72-hour incubation with 100 nmol/L vincristine, compared with euploid metaphases in untreated and HeLa-wt cells. F, Number of metaphase chromosomes from HeLa-wt, HeLa-MYC-S62A, and HeLa-MYC-T58A cells after 72-hour incubation with 10 and 100 nmol/L vincristine, unpaired t tests with Bonferroni corrections.

Figure 5.

S62A and T58A mutations impede vincristine-associated mitotic MYC degradation and mediate resistance to vincristine. A, Immunoblot of MYC in HeLa cells, after silencing endogenous MYC and expression of MYC-S62A and MYC-T58A. Accumulation in mitosis by DME for 16 hours, with or without vincristine in parallel. Mean values of three independent experiments, two-sided t test with Bonferroni correction. B, Viability of HeLa cells measured by MTS exposed to vincristine for 72 hours at the concentration indicated, mean values from three independent experiments, each run in triplicates. Two-sided t test, P value < 0.05 (***) according to Holm–Sidak method for HeLa-wt versus HeLa-S62A and HeLa-T58A for vincristine concentrations ≥ 10 nmol/L. C, Morphology of HeLa-MYC-S62A and HeLa-T58A and parental HeLa-wt cells incubated with vincristine with 10 and 20 nmol/L vincristine for 72 hours, washed and seeded in semisolid medium. Hela-S62A and HeLa-T58A cells formed colonies at day 7, whereas HeLa-wt cells showed few large, atypical cells after 10 nmol/L vincristine, and no viable cells after 20 nmol/L vincristine; scale bar, 100 μm. D, Cell viability by PI- and Hoechst33342 directly after 72-hour vincristine incubation. E, Hyperdiploid metaphases of HeLa-MYC-S62A and HeLa-MYC-T58A cells after 72-hour incubation with 100 nmol/L vincristine, compared with euploid metaphases in untreated and HeLa-wt cells. F, Number of metaphase chromosomes from HeLa-wt, HeLa-MYC-S62A, and HeLa-MYC-T58A cells after 72-hour incubation with 10 and 100 nmol/L vincristine, unpaired t tests with Bonferroni corrections.

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Postmitotic PI3K inhibition potentiates cytotoxic efficacy of vincristine

Having shown that MYC protein levels decrease during the M to G0–G1 transition, we searched for a mechanism to further deepen MYC loss at this point in the cell cycle. For unbiased evidence, we first mined the Genomics of Drug Sensitivity (GSDC) data for cell lines with MYC mutations. In this panel of 1,074 cell lines, we identified five cell lines with MYC missense mutations, involving aa138 in two cell lines (acute lymphoblastic leukemia MN-60, Burkitt lymphoma EB-3), and involving aa58 in three cell lines [B-cell lymphoma JM1, Burkitt lymphoma EB2, diffuse large cell B-cell lymphoma (DLBCL) Nu-DUL-1, http://www.cancerrxgene.org]. Consistent with our findings, the analysis revealed that the five cell lines with MYC mutations were resistant to drugs causing cell death in mitosis, but that they were particularly sensitive to the PI3K inhibitor idelalisib (Fig. 6A). The stability of MYC depends on GSK3β-mediated phosphorylation of MYC at Thr58 (11), which is negatively regulated by PI3K/AKT signaling during the G0–G1 phase of the cell cycle (36). Consistently, we found that the pan-PI3K inhibitor BKM120 depleted MYC in postmitotic HeLa cells within 2 to 4 hours at nanomolar concentrations, associated with low phosphorylation of AKT (Fig. 6B; Supplementary Fig. S6A), whereas at the same time interval, BKM120 did not deplete MYC levels in asynchronous cells (Fig. 6B).

Figure 6.

Postmitotic PI3K inhibition potentiates cytostatic efficacy of vincristine in vitro. A, Comparison of cell lines with and without mutations in the coding sequence of MYC in the GDSC panel of cancer cell lines (GDSC data; http://www.cancerrxgene.org). IC50 values from cell lines were compared for each drug by multivariate ANOVA. B, Immunoblot for MYC and AKT from HeLa cells following 4 hours of incubation with BKM120 in asynchronous or mitotic (M) HeLa cells. C, Immunoblot of MYC and AKT of A20 lymphoma cells after exposure to vincristine (20 nmol/L, 72 hours) and idelalisib (300 nmol/L, for 8 hours after removal of vincristine or DME) starting from equal cell number per condition, representative example of three experiments. D, MYC levels by immunoblot, mean values of three independent experiments for A20, and human BL cell lines DG75 and CA46 exposed to vincristine (20 nmol/L, 72 hours) and idelalisib (300 nmol/L, for 48 hours). Two-way ANOVA with Tukey correction for multiple comparisons. E, Expression and phosphorylation of myristoylated AKT1 in Hek293 cells (left plot) and reduced susceptibility to vincristine (20 nmol/L, 72 hours) in Hek293 and HeLa cells with expression of constitutively active AKT1; right plot, mean values of three independent experiments, two-sided t test with Bonferroni correction. F, Colony formation from P493-6 cells after sequential exposure to vincristine (20 nmol/L, 72 hours) and then idelalisib (100 nmol/L, continuously for 10 days), mean values ± SD of triplicates, P values of unpaired two-sided t test comparing colony formation 0 and 100 nmol/L idelalisib as indicated. G, Flow cytometric cell death of CA46, DG75, and OCI-Ly3 cells following exposure to vincristine 20 nmol/L for 72 hours and then idelalisib 300 nmol/L for 72 hours. H, Cell death of lymphoma cell lines incubated with 20 nmol/L vincristine alone (20 nmol/L, 72 hours/off-drug 72 hours) or the combination (20 nmol/L vincristine, 72 hours/300 nmol/L idelalisib, 72 hours). Inverse correlation of additional cell kill inversely vincristine susceptibility (Spearman's correlation coefficient r = -0.8107). I, Colony formation from cells lines after 72-hour exposure to vincristine (20 nmol/L), followed by continuous exposure to idelalisib (300 nmol/L) for 10 days. Mean ± SD of triplicates, representative examples of three independent experiments; two-sided paired t test; only data for cell lines with colony formation after exposure to vincristine shown.

Figure 6.

Postmitotic PI3K inhibition potentiates cytostatic efficacy of vincristine in vitro. A, Comparison of cell lines with and without mutations in the coding sequence of MYC in the GDSC panel of cancer cell lines (GDSC data; http://www.cancerrxgene.org). IC50 values from cell lines were compared for each drug by multivariate ANOVA. B, Immunoblot for MYC and AKT from HeLa cells following 4 hours of incubation with BKM120 in asynchronous or mitotic (M) HeLa cells. C, Immunoblot of MYC and AKT of A20 lymphoma cells after exposure to vincristine (20 nmol/L, 72 hours) and idelalisib (300 nmol/L, for 8 hours after removal of vincristine or DME) starting from equal cell number per condition, representative example of three experiments. D, MYC levels by immunoblot, mean values of three independent experiments for A20, and human BL cell lines DG75 and CA46 exposed to vincristine (20 nmol/L, 72 hours) and idelalisib (300 nmol/L, for 48 hours). Two-way ANOVA with Tukey correction for multiple comparisons. E, Expression and phosphorylation of myristoylated AKT1 in Hek293 cells (left plot) and reduced susceptibility to vincristine (20 nmol/L, 72 hours) in Hek293 and HeLa cells with expression of constitutively active AKT1; right plot, mean values of three independent experiments, two-sided t test with Bonferroni correction. F, Colony formation from P493-6 cells after sequential exposure to vincristine (20 nmol/L, 72 hours) and then idelalisib (100 nmol/L, continuously for 10 days), mean values ± SD of triplicates, P values of unpaired two-sided t test comparing colony formation 0 and 100 nmol/L idelalisib as indicated. G, Flow cytometric cell death of CA46, DG75, and OCI-Ly3 cells following exposure to vincristine 20 nmol/L for 72 hours and then idelalisib 300 nmol/L for 72 hours. H, Cell death of lymphoma cell lines incubated with 20 nmol/L vincristine alone (20 nmol/L, 72 hours/off-drug 72 hours) or the combination (20 nmol/L vincristine, 72 hours/300 nmol/L idelalisib, 72 hours). Inverse correlation of additional cell kill inversely vincristine susceptibility (Spearman's correlation coefficient r = -0.8107). I, Colony formation from cells lines after 72-hour exposure to vincristine (20 nmol/L), followed by continuous exposure to idelalisib (300 nmol/L) for 10 days. Mean ± SD of triplicates, representative examples of three independent experiments; two-sided paired t test; only data for cell lines with colony formation after exposure to vincristine shown.

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These data suggest a specific efficacy of PI3K inhibition against tumors, in which mutations protect mutant MYC from degradation. In addition, as PI3K inhibition and spindle disruption lower MYC levels through different mechanisms and at different time points during the cell cycle, we saw a rationale to test the effect of PI3K inhibition on MYC levels in cells exposed to vincristine during mitosis. We treated aggressive B-cell lymphoma cells first with vincristine followed by idelalisib. As expected, vincristine at 20 nmol/L for 72 hours abrogated mitotic MYC in murine A20 lymphoma cells and in a panel of human lymphoma cell lines (Fig. 6C, top plot; Supplementary Fig. S6B). Subsequent application of idelalisib then prevented postmitotic resurgence of MYC at 8 hours (Supplementary Fig. S6B) and pronounced after prolonged exposure at 48 hours, including the vincristine-resistant cell lines DG75 and CA46 (Fig. 6D; Supplementary Fig. S6C). Analyzing AKT phosphorylation as an event directly downstream of PI3K, idelalisib inhibited Ser473 phosphorylation in A20 cells when applied after vincristine (Fig. 6C). Vice versa we expected increased AKT activity to counteract the cytotoxic effect of vincristine attributable to MYC depletion. Thus, we expressed constitutively active, myristoylated AKT1 in HeLa and Hek293 cells (Fig. 6E, right plot), and we found that the expression of constitutive active AKT significantly increased the number of viable cells after exposure to vincristine (Fig. 6D, left plot).

Next, we addressed the impact of MYC expression on vincristine and idelalisib susceptibility in the P493-6–aggressive B-cell lymphoma model with doxycycline-sensitive ectopic MYC expression. Vincristine alone was highly cytotoxic, but subsequent exposure to idelalisib still significantly deepened the antitumor effects as measured in cell viability, death in mitosis and colony formation assays, and as expected, more efficiently in cells with high rather than low MYC status (Fig. 6F; Supplementary Fig. S7A and S7B). Extending the strategy to a panel of 16 Burkitt lymphoma and DLBCL cell lines confirmed the additional cytostatic effect of PI3K inhibition after prior vincristine (Fig. 6GI). Although in these experiments idelalisib was moderately effective as a single agent (mean cell death, 4.7%; range, 1%–11.7%), the efficacy of idelalisib increased substantially when applied after prior exposure to vincristine (mean cell death, 9.5%; range, 1.7%–19.7%). Overall, we observed an inverse correlation of single-agent vincristine cytotoxicity and combined vincristine/idelalisib cytotoxicity (Fig. 6H). Importantly, idelalisib also reduced the clonogenic growth from cells that survived vincristine exposure (Fig. 6I), supporting the notion that PI3K inhibition exerts its cytostatic effects predominantly in the postmitotic phase. The sequence of drug exposure was critical for the observed lethality of combined idelalisib and vincristine: when P493-6 cells were incubated with idelalisib before exposure to vincristine, the cells were arrested in G0–G1, resulting in reduced vincristine-mediated cytotoxicity and significantly increased CFU activity (Supplementary Fig. S7C–S7E). Finally, we applied the concept of sequential vincristine and PI3K inhibition in vivo in two murine-aggressive B-cell lymphoma models. We used the A20 B-lymphoblastic lymphoma that truly mimicked the behavior of human aggressive B-cell lymphoma in the in vitro studies, as well as the novel TA model, which had spontaneously developed in a cancer-susceptible mouse model and was passaged as a syngeneic tumor model in vivo only (Supplementary Fig. S8). Syngeneically implanted tumors were allowed to grow to 5 mm in diameter. Vincristine was dosed weekly, followed by PI3K inhibitor daily, starting 8 hours after vincristine. The PI3K inhibitor was paused for 48 hours prior to the next weekly dose of vincristine to allow for wash-out (Fig. 7A). As the pharmacokinetics of idelalisib prevent its use in mice, we employed the pan-PI3K inhibitor BKM120, and the alpha- and delta-preferential PI3K inhibitor copanlisib, both under clinical evaluation in lymphoma (37, 38). Consistent with the findings in vitro, sequential vincristine and BKM120 reduced MYC protein levels in the tumors and induced tumor necrosis (Fig. 7B), whereas neither vincristine nor the PI3K inhibitor, when given as single agents, was effective against the fast growth of these lymphomas. Sequential treatment significantly delayed tumor growth in both tumor models (Fig. 7C and E). In histologic analysis, tumor regressions induced by sequential vincristine and the PI3K inhibitors were associated with a marked reduction of cellularity, leaving only few interspersed tumor cells in the stroma (Fig. 7D and F).

Figure 7.

Postmitotic PI3K inhibition potentiates cytostatic efficacy of vincristine against aggressive murine B-cell lymphoma in vivo. A, Design of repeated combined tumor treatment with vincristine and PI3K inhibitor. Vincristine was given weekly and the PI3K inhibitor in between, starting 8 hours after vincristine injection and pausing 48 hours before next vincristine injection; time points of histology in A marked by corresponding stars. B, Expression of MYC in the TA murine-aggressive B-cell lymphoma model following short-term exposure to vincristine, PI3K inhibitor BKM120, and the combination of vincristine and BKM120. Samples obtained from tumor-bearing mice that had received no treatment (black star in A), 40-hour treatment with BKM120 (2 doses BKM120, 24 hours after start of BKM120), one dose of vincristine (72 hours after injection, red star in A), and the combination (one dose vincristine and three doses of BKM120, 40 hours after first BKM120, blue star in A); DAB staining with anti-MYC MoAb Y69, scale bar, 10 μm. C and E, Cytostatic efficacy of sequential vincristine/BKM120 and vincristine/copanlisib against murine-aggressive B-cell lymphomas TA and A20, respectively. Treatment groups were compared by two-way ANOVA tests followed by Tukey correction for multiple comparisons. D and F, Tumor histology at day 20 of treatment for TA tumors and day 12 for A20 tumors (hematoxylin and eosin stain; scale bars, 100 μm and 10 μm left and right columns, respectively).

Figure 7.

Postmitotic PI3K inhibition potentiates cytostatic efficacy of vincristine against aggressive murine B-cell lymphoma in vivo. A, Design of repeated combined tumor treatment with vincristine and PI3K inhibitor. Vincristine was given weekly and the PI3K inhibitor in between, starting 8 hours after vincristine injection and pausing 48 hours before next vincristine injection; time points of histology in A marked by corresponding stars. B, Expression of MYC in the TA murine-aggressive B-cell lymphoma model following short-term exposure to vincristine, PI3K inhibitor BKM120, and the combination of vincristine and BKM120. Samples obtained from tumor-bearing mice that had received no treatment (black star in A), 40-hour treatment with BKM120 (2 doses BKM120, 24 hours after start of BKM120), one dose of vincristine (72 hours after injection, red star in A), and the combination (one dose vincristine and three doses of BKM120, 40 hours after first BKM120, blue star in A); DAB staining with anti-MYC MoAb Y69, scale bar, 10 μm. C and E, Cytostatic efficacy of sequential vincristine/BKM120 and vincristine/copanlisib against murine-aggressive B-cell lymphomas TA and A20, respectively. Treatment groups were compared by two-way ANOVA tests followed by Tukey correction for multiple comparisons. D and F, Tumor histology at day 20 of treatment for TA tumors and day 12 for A20 tumors (hematoxylin and eosin stain; scale bars, 100 μm and 10 μm left and right columns, respectively).

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In summary, PI3K inhibition further depletes MYC protein immediately following mitosis contributing to the eradication of persistent cells that can overcome the G2–M arrest induced by vincristine. Hence, PI3K inhibition may be particularly active against aggressive lymphoma when administered after vincristine.

Binding of MYC to the mitotic spindle

Our key finding is that MYC, nonphosphorylated at Ser62, binds to the polymerized tubulin of the mitotic spindle, which may allow transfer of functional MYC to the daughter cells. Although MYC degradation during mitosis may provide a mechanism for transcriptional reset for the nascent cells on the one hand (18), tubule-mediated transmission of MYC would provide a scaffold for regulating the transfer of MYC to the new cells. Although we have no exact data on the amount of MYC at the spindle reservoir, the principle that cytoskeletal structures serve as docking sites from which an essential protein can be mobilized in its active form had been shown for transcription factors such as the release of NF-kB through microtubule depolymerization (39), or metabolic enzymes, such as aldolase that reversibly attaches to the actin cytoskeleton (40). Noteworthy, although we gained evidence for binding of MYC to microtubules, others have found phosphorylation-sensitive binding of MYC family members to other proteins, such as to the tumor suppressor BIN1(28), the binding of N-MYC to the kinase Aurora-A in neuroblastoma cells (41) or the binding of Ser62-phosphorylated MYC to nuclear lamins (42) with comparable affinities of the binding regions. The complex formation with Aurora-A protects N-MYC from phosphorylation and degradation in the G2–M phase, and it serves a function similar to the role described for tubulin here, but it may differ in the subcellular localization: Aurora-A primarily localizes with the centrosomes and extends to the spindle proximal to the poles only (41). Several serine kinases were described to phosphorylate MYC at the priming Ser62, including MAPK family members, polo-like kinase 1, calcium/calmodulin-dependent kinase IIg, and dual specificity tyrosine phosphorylation–regulated kinase 2 (11, 13, 43–45). In mitosis, turnover of N-MYC was found increased in cerebellar neural precursor cells, and phosphorylation of N-MYC at Ser54 required the CDK1 complex for Ser54 phosphorylation (18). Our data are consistent with Ser62 phosphorylation in mitosis acting as priming event for MYC degradation. Given the ambivalent role of Ser62 phosphorylation for both stabilization and degradation of MYC (8, 10–12), a scenario arises, in which several proteins beyond tubulin may compete for binding to the MB1 box and may thus govern the fate decision toward Thr58 phosphorylation–mediated degradation versus MYC protein stabilization. This mechanism may gain particular significance during symmetric and asymmetric cell divisions in stem and immune cells, where mitotic MYC regulation has already been identified as an important determinant of cell fate (19, 20). In mammalian development, mitotic MYC partition might operate in tandem with posttranslational regulation of GSK3ß, which phosphorylates MYC at Thr58 and which is inhibited as key downstream event in Wnt signaling. Importantly, inhibition of GSK3ß activity by Wnt-mediated posttranslational stabilization of proteins has been described also to culminate in mitosis (46).

Our data suggest that nonphosphorylated MYC binds to tubulin polymers, in line with previous results (23, 24, 47), whereas phosphorylated MYC loses its affinity to the tubulin polymer. Our data also show that tubulin polymer destabilization increases MYC destruction (Fig. 5), overall suggesting equilibrium of mitotic microtubule dynamics and the dynamics of MYC phosphorylation and decay in mitosis. This equilibrium is modulated by substances interfering with microtubule stability, e.g., by vincristine. Vinca-alkaloids bind to tubulin-beta within a central domain close to the GTP-binding site of the protein (48). As demonstrated for vinblastine and colchicine, the alkaloids create a wedge at the interdimer interface within the microtubule lattice and bend the protofilament growth axis (49), shifting the microtubule assembly/disassembly cycle toward depolymerization. The putative binding site of MYC at the plus end interface of the alpha/beta heterodimer might infer that the polymer formation affects—as we speculate—the binding of MYC to tubulin. However, at this point, we cannot exclude that tubulin dimers also bind to MYC at low affinity. Further characterizing the MYC/tubulin binding sites might open an avenue to design drugs either to stabilize MYC for use in regenerative medicine or to deplete MYC to fight cancer.

The MYC–microtubule interaction explains resistance to vinca-alkaloids

Perhaps the most immediate implications of our findings concern the mitotic MYC repartition in cancer cells: Mitotic depletion of MYC by vinca-alkaloids adds to explain the eminent efficacy of these drugs against MYC-driven and/or MYC-addicted tumors such as aggressive lymphatic neoplasms (Burkitt and Hodgkin's lymphoma, non-Hodgkin lymphoma) for MYC and neuroblastoma for N-MYC (50), and lends credence to the efficacy of approaches that use supplemental or intensified applications of vinca-alkaloids (51, 52).

Burkitt-type aggressive lymphoma is characterized by MYC activation based on chromosome 8;14 translocation and production of a highly active MYC–IgH fusion protein (53). In addition, MYC mutations in the MB1 region are a recurrent event in Burkitt lymphoma tumors (7, 54, 55), which, according to our data, would cause persistence of phosphorylation-deficient MYC through mitosis resulting in reduced susceptibility to vincristine.

Consistent with this reasoning, recent next-generation sequencing (NGS) analyses in another MYC-driven lymphoma, DLBCL, documented poor treatment outcomes specifically for those patients whose tumor carried a MYC-Thr58 mutation and who received a vincristine-containing regimen (56, 57). At this point, it remains open whether the poor outcome associated with MYC-Thr58 mutations is due to a direct growth advantage for tumor cells with high levels of postmitotic MYC, or indirectly linked to perturbation in chromosome segregation we discovered when cells with this mutation underwent DNA replication without cell division (Fig. 5). In line with these findings, elevated MYC levels have previously been recognized to cause chromosomal instability (58), which represents a pivotal mechanism in the progression and clonal evolution of tumors (59). Notably, the hotspot of MBI mutations found in clinical series extends from amino acid 56 through 63, which includes—but is not exclusive to—the Ser62 and Thr58 phosphorylation sites (55). It remains to analyze, however, whether amino acid exchanges other than Ser62 and Thr58 are relevant for the interaction of MYC with tubulin.

The dependence of daughter cells on inherited MYC is a unique opportunity for cancer treatment

Our observations identify the early postmitotic phase as a window for efficient tumor cell targeting. Suppressing of MYC activity in G1 phase through PI3K inhibition was linked with increased tumor cell death and strong impairment of clonogenic growth from surviving cells, consistent with previous reports that link PI3K signaling and MYC transcriptional activity to cell-cycle entry in G1 (60). The cooperation of PI3K and MYC is dependent on MYC stabilization via GSK3ß inhibition (61) and AKT-mediated phosphorylation of FoxO proteins (62). This cooperation is already active in Burkitt lymphoma tumorigenesis in mice, in which combining constitutive c-Myc expression and PI3K activity in germinal center B cells of the mouse led to Burkitt lymphoma–like tumors (63). Consistently in human Burkitt lymphoma disease, TCF3 mutations activating the PI3K pathway were found to occur at high frequency (54). Although the in vivo interactions of PI3K inhibition and vincristine certainly deserve further dissection, the data in this article support a role of PI3K activity for MYC stability through AKT activation as a potential treatment target. We show here that the oncogenic cooperation can be interrupted by applying G2–M arresting, MYC-depleting drugs first, followed by the PI3K inhibitor shortly thereafter. Repetitive rounds of sequential vincristine/PI3K inhibitor were highly effective in vivo, suggesting a strong potential for this treatment strategy in clinical trials for MYC-driven malignancies.

G.M. Wulf reports receiving other commercial research support from Merck & Co. No potential conflicts of interest were disclosed by the other authors.

Conception and design: C. Kiecke, E. Schäfer, R. Koch, M. de Oliveira Taveira, G.G. Wulf

Development of methodology: S. Becker, C. Kiecke, E. Schäfer, P. Trigo-Mourino, Z. Rydzynska, S. Dierks, H. Bastians, M. de Oliveira Taveira, G.G. Wulf

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Becker, C. Kiecke, E. Schäfer, U. Sinzig, P. Trigo-Mourino, C. Griesinger, R. Koch, F. von Bonin, H. Bohnenberger, J. Flach, S. Dierks, B. Maruschak, K. Bojarczuk, M. de Oliveira Taveira, L. Trümper, G.M. Wulf, G.G. Wulf

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Becker, C. Kiecke, E. Schäfer, L. Deuper, P. Trigo-Mourino, C. Griesinger, R. Koch, B. Chapuy, V. Venkataramani, A. Leha, S. Dierks, B. Maruschak, M. de Oliveira Taveira, G.M. Wulf, G.G. Wulf

Writing, review, and/or revision of the manuscript: C. Kiecke, E. Schäfer, P. Trigo-Mourino, R. Koch, B. Chapuy, D. Kube, K. Bojarczuk, M. de Oliveira Taveira, L. Trümper, G.M. Wulf, G.G. Wulf

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Becker, E. Schäfer, L. Deuper, B. Chapuy, F. von Bonin, L. Trümper, G.G. Wulf

Study supervision: C. Griesinger, G.G. Wulf

Others (technician): F. von Bonin

Others (selected experiments and data interpretation): D. Kube

This work was supported by the University Medicine Goettingen (Forschungsfoerderung to E. Schäfer) and the Goettinger Gesellschaft zur Unterstuetzung der Krebsforschung e.V (to G.G. Wulf). P. Trigo-Mourino acknowledges the Humboldt Foundation for a postdoctoral research fellowship.

FACS and cell sorting were performed in the Flow Cytometry Core Facility at the University of Goettingen. We thank A. Sands for linguistic editing.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Sheiness
DK
,
Hughes
SH
,
Varmus
HE
,
Stubblefield
E
,
Bishop
JM
. 
The vertebrate homolog of the putative transforming gene of avian myelocytomatosis virus: characteristics of the DNA locus and its RNA transcript
.
Virology
1980
;
105
:
415
24
.
2.
Dalla-Favera
R
,
Gelmann
EP
,
Martinotti
S
,
Franchini
G
,
Papas
TS
,
Gallo
RC
, et al
Cloning and characterization of different human sequences related to the onc gene (v-myc) of avian myelocytomatosis virus (MC29)
.
Proc Natl Acad Sci U S A
1982
;
79
:
6497
501
.
3.
Trumpp
A
,
Refaeli
Y
,
Oskarsson
T
,
Gasser
S
,
Murphy
M
,
Martin
GR
, et al
c-Myc regulates mammalian body size by controlling cell number but not cell size
.
Nature
2001
;
414
:
768
73
.
4.
Conacci-Sorrell
M
,
McFerrin
L
,
Eisenman
RN
. 
An overview of MYC and its interactome
.
Cold Spring Harb Perspect Med
2014
;
4
:
a014357
.
5.
Hann
SR
,
Eisenman
RN
. 
Proteins encoded by the human c-myc oncogene: differential expression in neoplastic cells
.
Mol Cell Biol
1984
;
4
:
2486
97
.
6.
Gregory
MA
,
Hann
SR
. 
c-Myc proteolysis by the ubiquitin-proteasome pathway: stabilization of c-Myc in Burkitt's lymphoma cells
.
Mol Cell Biol
2000
;
20
:
2423
35
.
7.
Malempati
S
,
Tibbitts
D
,
Cunningham
M
,
Akkari
Y
,
Olson
S
,
Fan
G
, et al
Aberrant stabilization of c-Myc protein in some lymphoblastic leukemias
.
Leukemia
2006
;
20
:
1572
81
.
8.
Luscher
B
,
Eisenman
RN
. 
Mitosis-specific phosphorylation of the nuclear oncoproteins Myc and Myb
.
J Cell Biol
1992
;
118
:
775
84
.
9.
Lutterbach
B
,
Hann
SR
. 
Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis
.
Mol Cell Biol
1994
;
14
:
5510
22
.
10.
Pulverer
BJ
,
Fisher
C
,
Vousden
K
,
Littlewood
T
,
Evan
G
,
Woodgett
JR
. 
Site-specific modulation of c-Myc cotransformation by residues phosphorylated in vivo
.
Oncogene
1994
;
9
:
59
70
.
11.
Sears
R
,
Nuckolls
F
,
Haura
E
,
Taya
Y
,
Tamai
K
,
Nevins
JR
. 
Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability
.
Genes Dev
2000
;
14
:
2501
14
.
12.
Farrell
AS
,
Sears
RC
. 
MYC degradation
.
Cold Spring Harb Perspect Med
2014
;
4
:
pii:
a014365
.
13.
Gu
Y
,
Zhang
J
,
Ma
X
,
Kim
BW
,
Wang
H
,
Li
J
, et al
Stabilization of the c-Myc protein by CAMKIIgamma promotes T cell lymphoma
.
Cancer Cell
2017
;
32
:
115
28 e7
.
14.
Salghetti
SE
,
Kim
SY
,
Tansey
WP
. 
Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc
.
EMBO J
1999
;
18
:
717
26
.
15.
Welcker
M
,
Orian
A
,
Jin
J
,
Grim
JE
,
Harper
JW
,
Eisenman
RN
, et al
The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation
.
Proc Natl Acad Sci U S A
2004
;
101
:
9085
90
.
16.
Kenney
AM
,
Cole
MD
,
Rowitch
DH
. 
Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors
.
Development
2003
;
130
:
15
28
.
17.
Hydbring
P
,
Bahram
F
,
Su
Y
,
Tronnersjo
S
,
Hogstrand
K
,
von der Lehr
N
, et al
Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation
.
Proc Natl Acad Sci U S A
2010
;
107
:
58
63
.
18.
Sjostrom
SK
,
Finn
G
,
Hahn
WC
,
Rowitch
DH
,
Kenney
AM
. 
The Cdk1 complex plays a prime role in regulating N-myc phosphorylation and turnover in neural precursors
.
Dev Cell
2005
;
9
:
327
38
.
19.
Topham
C
,
Tighe
A
,
Ly
P
,
Bennett
A
,
Sloss
O
,
Nelson
L
, et al
MYC is a major determinant of mitotic cell fate
.
Cancer Cell
2015
;
28
:
129
40
.
20.
Verbist
KC
,
Guy
CS
,
Milasta
S
,
Liedmann
S
,
Kaminski
MM
,
Wang
R
, et al
Metabolic maintenance of cell asymmetry following division in activated T lymphocytes
.
Nature
2016
;
532
:
389
93
.
21.
Rottenberg
S
,
Nygren
AO
,
Pajic
M
,
van Leeuwen
FW
,
van der Heijden
I
,
van de Wetering
K
, et al
Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer
.
Proc Natl Acad Sci U S A
2007
;
104
:
12117
22
.
22.
Stolz
A
,
Ertych
N
,
Bastians
H
. 
A phenotypic screen identifies microtubule plus end assembly regulators that can function in mitotic spindle orientation
.
Cell Cycle
2015
;
14
:
827
37
.
23.
Alexandrova
N
,
Niklinski
J
,
Bliskovsky
V
,
Otterson
GA
,
Blake
M
,
Kaye
FJ
, et al
The N-terminal domain of c-Myc associates with alpha-tubulin and microtubules in vivo and in vitro
.
Mol Cell Biol
1995
;
15
:
5188
95
.
24.
Niklinski
J
,
Claassen
G
,
Meyers
C
,
Gregory
MA
,
Allegra
CJ
,
Kaye
FJ
, et al
Disruption of Myc-tubulin interaction by hyperphosphorylation of c-Myc during mitosis or by constitutive hyperphosphorylation of mutant c-Myc in Burkitt's lymphoma
.
Mol Cell Biol
2000
;
20
:
5276
84
.
25.
Vallee
RB
. 
On the use of heat stability as a criterion for the identification of microtubule associated proteins (MAPs)
.
Biochem Biophys Res Commun
1985
;
133
:
128
33
.
26.
Canales
A
,
Nieto
L
,
Rodriguez-Salarichs
J
,
Sanchez-Murcia
PA
,
Coderch
C
,
Cortes-Cabrera
A
, et al
Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches
.
ACS Chem Biol
2014
;
9
:
1033
43
.
27.
Kadavath
H
,
Cabrales Fontela
Y
,
Jaremko
M
,
Jaremko
L
,
Overkamp
K
,
Biernat
J
, et al
The binding mode of a tau peptide with tubulin
.
Angew Chem
2018
;
57
:
3246
50
.
28.
Pineda-Lucena
A
,
Ho
CS
,
Mao
DY
,
Sheng
Y
,
Laister
RC
,
Muhandiram
R
, et al
A structure-based model of the c-Myc/Bin1 protein interaction shows alternative splicing of Bin1 and c-Myc phosphorylation are key binding determinants
.
J Mol Biol
2005
;
351
:
182
94
.
29.
Richards
MW
,
Burgess
SG
,
Poon
E
,
Carstensen
A
,
Eilers
M
,
Chesler
L
, et al
Structural basis of N-Myc binding by Aurora-A and its destabilization by kinase inhibitors
.
Proc Natl Acad Sci U S A
2016
;
113
:
13726
31
.
30.
Neron
B
,
Menager
H
,
Maufrais
C
,
Joly
N
,
Maupetit
J
,
Letort
S
, et al
Mobyle: a new full web bioinformatics framework
.
Bioinformatics
2009
;
25
:
3005
11
.
31.
Nogales
E
,
Wolf
SG
,
Downing
KH
. 
Structure of the alpha beta tubulin dimer by electron crystallography
.
Nature
1998
;
391
:
199
203
.
32.
Noble
RL
,
Beer
CT
,
Cutts
JH
. 
Role of chance observations in chemotherapy: vinca rosea
.
Ann N Y Acad Sci
1958
;
76
:
882
94
.
33.
Schiff
PB
,
Horwitz
SB
. 
Taxol stabilizes microtubules in mouse fibroblast cells
.
Proc Natl Acad Sci U S A
1980
;
77
:
1561
5
.
34.
Dumontet
C
,
Jordan
MA
. 
Microtubule-binding agents: a dynamic field of cancer therapeutics
.
Nat Rev Drug Discovery
2010
;
9
:
790
803
.
35.
Frei
E
 3rd
,
Whang
J
,
Scoggins
RB
,
Vanscott
EJ
,
Rall
DP
,
Ben
M
. 
The stathmokinetic effect of vincristime
.
Cancer Res
1964
;
24
:
1918
25
.
36.
Liang
J
,
Slingerland
JM
. 
Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression
.
Cell Cycle
2003
;
2
:
339
45
.
37.
Paul
J
,
Soujon
M
,
Wengner
AM
,
Zitzmann-Kolbe
S
,
Sturz
A
,
Haike
K
, et al
Simultaneous inhibition of PI3Kdelta and PI3Kalpha induces ABC-DLBCL regression by blocking BCR-dependent and -independent activation of NF-kappaB and AKT
.
Cancer Cell
2017
;
31
:
64
78
.
38.
Younes
A
,
Salles
G
,
Martinelli
G
,
Bociek
RG
,
Caballero Barrigon
D
,
Gonzalez Barca
E
, et al
Pan-phosphatidylinositol 3-kinase inhibition with buparlisib in patients with relapsed and refractory non-Hodgkin lymphoma
.
Haematologica
2017
;
102
:
2104
12
.
39.
Rosette
C
,
Karin
M
. 
Cytoskeletal control of gene expression: depolymerization of microtubules activates NF-kappa B
.
J Cell Biol
1995
;
128
:
1111
9
.
40.
Hu
H
,
Juvekar
A
,
Lyssiotis
CA
,
Lien
EC
,
Albeck
JG
,
Oh
D
, et al
Phosphoinositide 3-kinase regulates glycolysis through mobilization of aldolase from the actin cytoskeleton
.
Cell
2016
;
164
:
433
46
.
41.
Otto
T
,
Horn
S
,
Brockmann
M
,
Eilers
U
,
Schuttrumpf
L
,
Popov
N
, et al
Stabilization of N-Myc is a critical function of Aurora A in human neuroblastoma
.
Cancer Cell
2009
;
15
:
67
78
.
42.
Myant
K
,
Qiao
X
,
Halonen
T
,
Come
C
,
Laine
A
,
Janghorban
M
, et al
Serine 62-phosphorylated MYC associates with nuclear lamins and its regulation by CIP2A is essential for regenerative proliferation
.
Cell Rep
2015
;
12
:
1019
31
.
43.
Tan
J
,
Li
Z
,
Lee
PL
,
Guan
P
,
Aau
MY
,
Lee
ST
, et al
PDK1 signaling toward PLK1-MYC activation confers oncogenic transformation, tumor-initiating cell activation, and resistance to mTOR-targeted therapy
.
Cancer Discov
2013
;
3
:
1156
71
.
44.
Taira
N
,
Mimoto
R
,
Kurata
M
,
Yamaguchi
T
,
Kitagawa
M
,
Miki
Y
, et al
DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells
.
J Clin Invest
2012
;
122
:
859
72
.
45.
Lutterbach
B
,
Hann
SR
. 
c-Myc transactivation domain-associated kinases: questionable role for map kinases in c-Myc phosphorylation
.
J Cell Biochem
1999
;
72
:
483
91
.
46.
Acebron
SP
,
Karaulanov
E
,
Berger
BS
,
Huang
YL
,
Niehrs
C
. 
Mitotic wnt signaling promotes protein stabilization and regulates cell size
.
Mol Cell
2014
;
54
:
663
74
.
47.
Koch
HB
,
Zhang
R
,
Verdoodt
B
,
Bailey
A
,
Zhang
CD
,
Yates
JR
 3rd
, et al
Large-scale identification of c-MYC-associated proteins using a combined TAP/MudPIT approach
.
Cell Cycle
2007
;
6
:
205
17
.
48.
Rai
SS
,
Wolff
J
. 
Localization of the vinblastine-binding site on beta-tubulin
.
J Biol Chem
1996
;
271
:
14707
11
.
49.
Stanton
RA
,
Gernert
KM
,
Nettles
JH
,
Aneja
R
. 
Drugs that target dynamic microtubules: a new molecular perspective
.
Med Res Rev
2011
;
31
:
443
81
.
50.
Jordan
MA
,
Thrower
D
,
Wilson
L
. 
Mechanism of inhibition of cell proliferation by vinca alkaloids
.
Cancer Res
1991
;
51
:
2212
22
.
51.
Wilson
WH
,
Grossbard
ML
,
Pittaluga
S
,
Cole
D
,
Pearson
D
,
Drbohlav
N
, et al
Dose-adjusted EPOCH chemotherapy for untreated large B-cell lymphomas: a pharmacodynamic approach with high efficacy
.
Blood
2002
;
99
:
2685
93
.
52.
O'Brien
S
,
Schiller
G
,
Lister
J
,
Damon
L
,
Goldberg
S
,
Aulitzky
W
, et al
High-dose vincristine sulfate liposome injection for advanced, relapsed, and refractory adult Philadelphia chromosome-negative acute lymphoblastic leukemia
.
J Clin Oncol
2013
;
31
:
676
83
.
53.
Dalla-Favera
R
,
Bregni
M
,
Erikson
J
,
Patterson
D
,
Gallo
RC
,
Croce
CM
. 
Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells
.
Proc Natl Acad Sci U S A
1982
;
79
:
7824
7
.
54.
Schmitz
R
,
Young
RM
,
Ceribelli
M
,
Jhavar
S
,
Xiao
W
,
Zhang
M
, et al
Burkitt lymphoma pathogenesis and therapeutic targets from structural and functional genomics
.
Nature
2012
;
490
:
116
20
.
55.
Lopez
C
,
Kleinheinz
K
,
Aukema
SM
,
Rohde
M
,
Bernhart
SH
,
Hubschmann
D
, et al
Genomic and transcriptomic changes complement each other in the pathogenesis of sporadic burkitt lymphoma
.
Nat Commun
2019
;
10
:
1459
.
56.
Xu-Monette
ZY
,
Deng
Q
,
Manyam
GC
,
Tzankov
A
,
Li
L
,
Xia
Y
, et al
Clinical and biologic significance of MYC genetic mutations in de novo diffuse large B-cell lymphoma
.
Clin Cancer Res
2016
;
22
:
3593
605
.
57.
Reddy
A
,
Zhang
J
,
Davis
NS
,
Moffitt
AB
,
Love
CL
,
Waldrop
A
, et al
Genetic and functional drivers of diffuse large B cell lymphoma
.
Cell
2017
;
171
:
481
94 e15
.
58.
Li
Q
,
Dang
CV
. 
c-Myc overexpression uncouples DNA replication from mitosis
.
Mol Cell Biol
1999
;
19
:
5339
51
.
59.
Laughney
AM
,
Elizalde
S
,
Genovese
G
,
Bakhoum
SF
. 
Dynamics of tumor heterogeneity derived from clonal karyotypic evolution
.
Cell Rep
2015
;
12
:
809
20
.
60.
Cantley
LC
,
Neel
BG
. 
New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway
.
Proc Natl Acad Sci U S A
1999
;
96
:
4240
5
.
61.
Kumar
A
,
Marques
M
,
Carrera
AC
. 
Phosphoinositide 3-kinase activation in late G1 is required for c-Myc stabilization and S phase entry
.
Mol Cell Biol
2006
;
26
:
9116
25
.
62.
Bouchard
C
,
Marquardt
J
,
Bras
A
,
Medema
RH
,
Eilers
M
. 
Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins
.
EMBO J
2004
;
23
:
2830
40
.
63.
Sander
S
,
Calado
DP
,
Srinivasan
L
,
Kochert
K
,
Zhang
B
,
Rosolowski
M
, et al
Synergy between PI3K signaling and MYC in Burkitt lymphomagenesis
.
Cancer Cell
2012
;
22
:
167
79
.