Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kδ and CK1ε in hematological malignancies

c-Myc is a master transcription factor and one of the most frequently altered genes across a vast array of human cancers including diffuse large B-cell lymphoma (DLBCL),^1,2  and is thus an attractive therapeutic target.³  However, no direct inhibitor of c-Myc has been successfully developed for the treatment of any cancer. The c-Myc protein has a short half-life of <30 minutes,⁴  and the complex secondary structures in the 5′ untranslated region (UTR) of MYC messenger RNA (mRNA) make its translation highly dependent on the eukaryotic translation initiation factor 4F (eIF4F).^5,6  eIF4F exists as a complex composed of the eIF4E, eIF4A, and eIF4G subunits. eIF4E can be sequestered by eIF4E-binding protein 1 (4E-BP1), which acts as a “brake” for initiation of mRNA translation.⁷  Hyperphosphorylation of 4E-BP1, caused by upstream signals such as mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), leads to release of eIF4E from 4E-BP1, assembly of the eIF4F complex, and robust mRNA translation.^8-10  In keeping with these data, mTORC1 and dual mTORC1/mTORC2 inhibitors have been found to cause varied degrees of inhibition of 4E-BP1 phosphorylation and translation initiation for tumor-promoting genes.^11-17  However, the therapeutic effects of mTOR inhibition in c-Myc–driven cancer remain poorly understood.

A number of mTORC1 inhibitors have been approved for renal cell cancer, but they demonstrate limited activity in other cancers including DLBCL. The dual mTORC1/mTORC2 inhibitor MLN0128 was recently reported to exhibit no activity in lymphoma.¹⁴  These results suggest that most cancers, including lymphoma, likely use multiple signaling pathways to ensure robust translation, and therefore are able to bypass translational downregulation caused by mTOR inhibitors. As an example, casein kinase-1 ε (CK1ε) activates mRNA translation through phosphorylating 4E-BP1 at residues distinct from those responsive to mTOR.¹⁸  PI3K is also involved in phosphorylating 4E-BP1 independently of mTORC1.¹⁹  Furthermore, there is emerging evidence that the proteasome system is involved in the activation of mTORC1,^20,21  presumably through regulating the intracellular pool of amino acids.^22,23  Collectively, these data suggest that phosphorylation of 4E-BP1 orchestrates multiple upstream signals required for optimal translation initiation for dynamic proteins in high demand, such as c-Myc. We hypothesized that modulating 4E-BP1 using a multitargeting approach against PI3K, the proteasome, and CK1ε could be an effective strategy for silencing of c-Myc translation in aggressive c-Myc–dependent lymphoma.

Additional methods are described in supplemental Methods, available on the Blood Web site.

The cell lines were obtained from ATCC and grown in Iscove modified Dulbecco medium with 10% fetal calf serum. Carfilzomib, bortezomib, and idelalisib were purchased from Selleck. TGR-1202 was provided by TG Therapeutics.

Cytotoxicity was performed on cultured cells using the Cell Titer Glo assay, as previously described.²⁴ 

In vitro studies in cell lines were repeated twice, and those in primary patient samples and mice were done once. All cytotoxicity studies were done with triple replicas. The mean and the standard error of the mean were graphed or charted. Synergy was measured by excess over Bliss values.²⁵  For the in vivo studies, mice were randomized to different treatment cohorts. Statistical analysis of difference in tumor volume and tumor weight among the groups was evaluated using a 1-way analysis of variance followed by individual comparisons using least significant difference (equal variance assumed). All significance testing was done at the P < .05 level, protecting the family-wise error rate.

TGR-1202 has the core structure required for targeting phosphoinositide 3-kinase δ (PI3Kδ), as circled in Figure 1A. In a cell-free kinase assay based on detection of phosphatidylinositol (3,4,5)-trisphosphate, TGR-1202 potently inhibited PI3Kδ with a half maximal effective concentration (EC₅₀) of 22.2 nM (Figure 1B). The EC₅₀ values of TGR-1202 against the other isoforms of PI3K were substantially higher (supplemental Figure 1A), confirming TGR-1202 is a PI3Kδ inhibitor with a selectivity comparable to idelalisib,²⁶  which is a first-in-class approved PI3Kδ inhibitor. Next, human lymphoma and leukemia cell lines known for constitutively activated AKT were treated with TGR-1202 or the vehicle control for 4 hours. TGR-1202 inhibited phosphorylated AKT at Ser473 in a concentration-dependent manner (Figure 1C). At 1 μM, TGR-1202 reduced the phosphorylation of AKT by 43% to 87% in these cell lines. In a subcutaneous xenograft model of T-cell acute lymphoblastic leukemia (T-ALL) in NOD/SCID mice using the MOLT-4 cell line,²⁷  daily oral treatment with TGR-1202 at 150 mg/kg significantly shrank the tumors by day 25 (P < .001) (Figure 1D). In select lymphoma cell lines representing DLBCL (LY7), mantle cell lymphoma (MCL; Z-138), and cutaneous T-cell lymphoma (CTCL; H9), TGR-1202 was more active than idelalisib inhibiting the viability of lymphoma cells, based on measurement of adenosine triphosphate (ATP) in metabolically active and viable cells (Figure 1E). In other lymphoma cell lines, however, TGR-1202 and idelalisib demonstrated a comparably modest antilymphoma activity (supplemental Figure 1B).

Because PI3Kδ inhibitors as single agents demonstrated only limited activity in models of aggressive lymphoma, we explored whether PI3Kδ inhibitors (TGR-1202 and idelalisib) might synergize with proteasome inhibitors (carfilzomib and bortezomib) through synergistic repression of mTOR signaling. Four combination pairs were studied in the DLBCL cell line LY10, including TGR-1202 plus carfilzomib (TC), idelalisib plus carfilzomib (IC), TGR-1202 plus bortezomib (TB), and idelalisib plus bortezomib (IB). We adopted the Bliss additivism model,²⁵  which has been successfully used by the NCI-DREAM Drug Sensitivity Prediction Challenge, to predict drug:drug synergy. The model first calculates the expected inhibition for 2 drugs that are assumed to be additive, which is then used to calculate the excess over Bliss (EOB) value as an index of synergy. TC was clearly the most synergistic among the 4 combinations as shown by highly positive EOB values in the DLBCL cell line LY10 (Figure 2A; supplemental Figure 2A) and 11 other lymphoma cell lines representing DLBCL (LY1, SUDHL-4, SUDHL-2, and LY7), MCL (Jeko-1, Z138), T-ALL (PF-382, P12), CTCL (H9 and HH), and multiple myeloma (MM) (MM.1s) (Figure 2B; supplemental Figure 2B-C). TC was highly synergistic in primary lymphoma and leukemia cells isolated fresh from 4 patients with relapsed small lymphocytic lymphoma (SLL), treatment naive chronic lymphocytic leukemia (CLL), treatment naive blastoid MCL with p53 deletion, and marginal zone lymphoma with chromosome 17p deletion (Figure 2C; supplemental Figure 2C). The TC combination synergistically induced apoptosis, as measured by the cleavage of poly (ADP-ribose) polymerase and activation of caspase 3/7, in those cell lines and primary lymphoma and leukemia cells, whereas the IB and IC combinations were less active by these assays (Figure 2D; supplemental Figure 2D). TC was not toxic to normal peripheral mononuclear cells (supplemental Figure 2F).

Next, we investigated the combinations described in the previous paragraph and single agents for their differential effects on mTOR signaling, focusing on the mTORC1 substrate 4E-BP1 and a further downstream event, namely translation of c-Myc. The highly synergistic TC combination, but not IB or single agents, potently inhibited phosphorylation of 4E-BP1 and the protein level of c-Myc in cell lines representing DLBCL (LY10 and LY7), T-ALL (PF382), MM (H929), as well as primary SLL and CLL cells (Figure 3).

The reduction of the c-Myc protein level by TC in the cell lines described in the previous paragraphs and primary lymphoma cells was unlikely due to increased degradation of c-Myc because carfilzomib is a potent proteasome inhibitor, but rather could be due to inhibition of transcription or translation. Although the IB, TB, and IC combinations moderately reduced the protein level of c-Myc, TC markedly inhibited the expression of c-Myc in the LY10 cells (Figure 4A). Because none of the combinations decreased the mRNA level of c-Myc in LY10 or LY7 (Figure 4B), TC most likely reduced c-Myc protein level by inhibiting its translation. To further prove this hypothesis, we designed a bicistronic luciferase reporter (Figure 4C). Translation of Renilla luciferase (LucR) is regulated by the 5′ UTR of c-MYC and is dependent on eIF4F. In contrast, translation of firefly luciferase (LucF) is not eIF4F dependent as it has the Polio virus internal ribosome entry site (IRES). The relative efficiency of cap-dependent translation downstream of the 5′ UTR of c-MYC is measured by the ratio of LucR/LucF. The plasmid did transduce LY7 efficiently, but did not transfect LY10 and a number of other DLBCL cell lines. Figure 4D demonstrates that TC was significantly more potent than IB in LY7 in reducing the R:F Luc ratio (P = .0013), confirming c-Myc translation as a mechanistic target of TC.

To confirm that c-Myc downregulation is a mechanistic target of the TC combination, we subcloned the MYC open reading frame without the 5′ UTR, into the pcDNA3.1(+)IRES green fluorescent protein plasmid. As a result, translation of exogenous c-Myc is expected to be less dependent on eIF4F and less prone to inhibition by 4E-BP1. As shown in Figure 4E, the c-Myc–containing plasmid (M+) mildly increased the level of c-Myc protein compared with an empty vector (EV) in the LY7 DLBCL cells not exposed to the TC treatment. When treated with TC, the level of the endogenous c-Myc protein was potently reduced in the cells transduced with the EV. In contrast, the level of c-Myc remained at a high level in the cells with the Myc+ plasmid, confirming that lack of 5′ UTR of MYC in the plasmid conveyed resistance to translational downregulation of MYC by TC. Figure 4F demonstrates that LY7 cells with the Myc+ plasmid were significantly more resistant than those cells with the EV to TC. To investigate whether sequestering of eIF4E by hypophosphorylated 4E-BP1 was a potential mechanism of action by TC, we subcloned the complementary DNA of eIF4E into the pCDH-GFP vector. For poorly understood reasons, the eIF4E plasmid was toxic to the DLBCL cell line LY7. We therefore stably transduced this plasmid and a control EV into the myeloma cell line H929, which has a well-characterized chromosomal translocation involving c-Myc.²⁸ Figure 4G demonstrated that in H929 cells untreated with TC, the eIF4E plasmid produced slightly more eIF4E and c-Myc than the EV. In H929 cells treated with TC, the eIF4E plasmid produced substantially more eIF4E and c-Myc proteins than the EV. Cells transfected with the eIF4E plasmid were significantly more resistant to TC than cells with the EV (Figure 4H). Collectively, these results indicate that the mechanism of the remarkable synergism observed with the TC combination is due to inhibition of 4E-BP1 phosphorylation and consequently downregulation of c-Myc translation.

To investigate the effects of TC on the c-Myc transcription program, gene expression profiling was performed by RNA sequencing in the DLBCL LY10 cells treated by the vehicle control, TGR-1202, idelalisib, carfilzomib, bortezomib, and the 4 combinations including TC, TB, IC, and IB for 24 hours. We calculated fold change for each gene and the P value in the combinations vs each of the single-drug exposures. Next, we combined the 2 P values to calculate a combined Z score, which were used to rank list the genes according to their upregulation or downregulation at the transcriptional level by the combinations. We then performed gene set enrichment analysis (GSEA) to evaluate the enrichment of c-Myc target genes using the annotated gene sets in the Molecular Signatures Database (MSigDB). Figure 5A demonstrates that in the LY10 cells treated by TC, the running enrichment score (RES) of 4 “canonical” Myc target gene sets (GS52, GS70, GS29, and GS32) reached their peak RES scores at the end of the gene list ranked from most upregulated to most downregulated, with normalized enrichment score (NES) < 0 and false discovery rate (FDR) q value (q-val) = 0. In contrast, most unrelated gene sets have higher NES and/or FDR values in the TC treated sample (supplemental Figure 3A). We independently validated that 2 of the c-Myc target genes, eIF4B and E2F1, were markedly reduced by the TC combination but not by any single agents or the IB combination in the DLBCL cells LY10 and LY7 (Figure 5B). In agreement, E2F gene sets (GS43, GS38, and GS22) were also enriched among the downregulated genes (NES < 0 and FDR q-val = 0) by TC more than any other combination treatments (supplemental Figure 3B). We conducted GSEA on all the Myc and E2F target gene sets, and found that the number of c-Myc and E2F1 gene sets downregulated by TC was significantly higher than those affected by IB (P = .014 for Myc and P < .00001 for E2F) (Figure 5C; supplemental Tables 1-2). Collectively, these results demonstrate that TGR-1202 and carfilzomib in combination synergistically and selectively silence the c-Myc and E2F transcription programs.

To understand why TGR-1202 was consistently superior to idelalisib when combined with carfilzomib, we compared the activity of TGR-1202 with 2 other PI3Kδ inhibitors, idelalisib and duvelisib/IPI-145, on a panel of 365 wild-type protein kinases using the kinome profiling platform from Reaction Biology (Malvern, PA). The PI3Kδ inhibitors were not active against this panel of protein kinases with 1 exception: at 1 μM, TGR-1202 inhibited 60% of the activity of CK1ε, which was not observed with idelalisib or duvelisib (Figure 6A; supplemental Table 3).

Remarkably, TGR-1202 and the selective CK1ε inhibitor PF4800567²⁹  share a similar overall architecture with a central pyrazolopyrimidine amine (CPA) moiety substituted at the same positions 7 and 9 (Figure 6B). A previous x-ray crystallography study of PF4800567 has confirmed that the CPA moiety plays a key role in the binding of the inhibitor to CK1ε as it establishes 2 hydrogen bonds with the hinge region of CK1ε (supplemental Figure 4A).³⁰  In this orientation, the chlorobenzene moiety of PF4800567 (substituted at position 7), occupies a hydrophobic pocket deeper in the protein (Figure 6C). In silico docking of TGR-1202 into the ATP pocket of CK1ε resulted in top scoring (best docking score, −9.3) binding modes very consistent with that of PF4800567, with the CPA moiety superposing very well and establishing the exact same hydrogen bonds (Figure 6C-D). Importantly, the favorable docking scores obtained for these virtual binding modes reveal that the hydrophobic pocket reached by the chlorobenzene moiety for PF4800567 can favorably accommodate the somewhat larger corresponding moiety in TGR-1202. In contrast, although idelalisib contains an adenine moiety that is reminiscent of the CPA moiety shared by PF4800567 and TGR-1202, the potential hydrogen bond donors and acceptors are distributed very differently (supplemental Figure 4B). Consistently, in silico docking of idelalisib fails to identify high-scoring binding modes (best docking score, −3.8) into the CK1ε ATP pocket.

Based on this structural insight, we synthesized 2 TGR-1202 analogs, CUX-03173 and CUX-03166, which differ by 1 extra methyl group on CUX-03166 (supplemental Figure 4C). Consistent with its high chemical similarity with TGR-1202, in silico docking of CUX-03173 results in a top-binding pose very close to that of TGR-1202 (supplemental Figure 4D-E). In contrast, in silico docking of CUX-03166 in CK1ε results in poses with significantly worse docking scores than CUX-03173 and TGR-1202, as the floor of the ATP-binding pocket leaves insufficient room for the methyl group on CUX-03166.

We experimentally determined the CK1ε–inhibiting activity of the previously described compounds using the ADP-Glo Kinase Assay kit and recombinant CK1ε expressed by baculovirus in Sf9 insect cells. PF4800567 was highly potent against CK1ε with an EC₅₀ of 7.4 nM (Figure 6E; supplemental Figure 4F), consistent with the previous report.²⁹  TGR-1202 was active against CK1ε, with an EC₅₀ value of 6.0 μM. The EC₅₀ for CUX-03173 was 9.4 μM. In contrast, idelalisib or CUX-03166 did not reach 50% inhibition even at 50 μM. Next, we directly tested the effects of TGR-1202 on intracellular CK1ε by examining its effect on the autophosphorylation of the CK1ε C-terminal regulatory domain.^31-33  Autophosphorylation is inhibited or stimulated by CK1ε inhibitors or phosphatase inhibitors such as calyculin A, respectively. In the negative control not treated with any of the tested kinase inhibitors, calyculin A produced time-dependent autophosphorylation of CK1ε, as shown by upshifting and dimming of the CK1ε band in the DLBCL cell line LY7 (Figure 6F). In samples treated by PF4800567 (1 μM), TGR-1202 and CUX-03173 (10-25 μM) the upshifting of the CK1ε band was delayed and reduced (Figure 6F; supplemental Figure 4G). Idelalisib at 25 μM remained inactive by this assay (supplemental Figure 4G). These results demonstrate that among the studied PI3Kδ inhibitors, TGR-1202 is uniquely characterized with structural features suitable for targeting CK1ε in lymphoma cells.

To establish that targeting CK1ε is required for optimal treatment of c-Myc–dependent lymphoma, we first compared the effects of the dual PI3Kδ/CK1ε inhibitor TGR-1202, selective PI3Kδ inhibitor idelalisib, and selective CK1ε inhibitor PF4800567 on c-Myc and 4E-BP1. Figure 7A demonstrates that, at concentrations ranging from 15 to 50 μM, TGR-1202 was more potent than idelalisib and PF4800567 at repressing the expression of c-Myc in the DLBCL cell line LY7. Importantly, lymphoma cells transduced with the c-Myc–overexpressing plasmid (M+) described in Figure 4E were significantly more resistant to TGR-1202 than the cells with the EV (Figure 7B; supplemental Figure 5A). To determine whether the reduction of c-Myc protein level by TGR-1202 was due to downregulated c-Myc translation, we performed the reporter assay as described in Figure 4C-D. The ratio of LucR:LucF was reduced by >50% by TGR-1202 at 15 μM but not by idelalisib or PF4800567 (P < .001) (Figure 7C). Interestingly, combining the selective PI3Kδ inhibitor idelalisib and selective CK1ε inhibitor PF4800567, both at 25 μM, reproduced the potent inhibition of 4E-BP1 phosphorylation and c-Myc protein level caused by the dual PI3Kδ/CK1ε inhibitor TGR-1202 at 25 μM in LY7 cells (Figure 7D). These results indicate that cotargeting PI3Kδ and CK1ε produces synergistic inhibition of 4E-BP1 phosphorylation and Myc translation.

Next, we investigated whether knockdown of CK1ε could phenocopy TGR-1202 in the combination of idelalisib and carfilzomib, which was shown to be significantly less synergistic than TC (Figures 2A and 4A). CK1ε-targeting short hairpin RNA (shRNA) reduced the protein level of CK1ε by >80%, but had no effect on c-myc, in the DLBCL cell line LY7 (Figure 7E; supplemental Figure 5B). In LY7 cells with wild-type CK1ε, the combination of idelalisib and carfilzomib produced only mild inhibition of phosphorylation of 4E-BP1, as evidenced by a partial downward mobility shift of 4E-BP1. In contrast, in the cells with CK1ε knockdown, the combination of idelalisib and carfilzomib effectively inhibited phosphorylation of 4E-BP1, demonstrated by a complete mobility shift of 4E-BP1 and a substantial decrease in phosphorylated 4E-BP1 at T70. These results indicate that targeting CK1ε complements PI3Kδ inhibition in the setting of combination therapy with carfilzomib.

In a phase 1 clinical trial of TGR-1202 as a single agent (#NCT01767766), partial response was observed in 3 of 14 patients with DLBCL (Figure 7F-G; supplemental Figure 6A-B). In contrast, earlier studies of idelalisib did not observe any responses in 9 patients with DLBCL.³⁴  Furthermore, TGR-1202 was associated with very limited (2%) grade 3/4 diarrhea, compared with 13% seen with idelalisib in patients with similar clinical parameters.³⁵  These results suggest that dual targeting of PI3Kδ and CK1ε by TGR-1202 may explain in part the preliminary activity of TGR-1202 in treating DLBCL and its distinctly favorable adverse event profile.

For many years, c-Myc has been recognized as an “undruggable” target. More recently, BRD4 inhibitors have shown promise as a c-Myc–targeting therapy,³⁶  with a few of them having entered phase 1 clinical studies. However, their safety and efficacy remain to be proven. In the current work, we aimed to explore new therapeutic strategies for targeting c-Myc that could be rapidly validated in clinical studies. To that end, we have demonstrated that the combination of TGR-1202 and carfilzomib, inhibitors of PI3Kδ and proteasome, respectively, potently disrupts the 4E-BP1–eIF4F–c-Myc axis, leading to antitumor effects of TGR-1202 and carfilzomib in cell lines and primary cells representing broad subtypes of B- and T-cell lymphoma, leukemia, and MM. The unique synergism of TGR-1202 and carfilzomib is driven in part by the unique pharmacologic activities of TGR-1202 reported in the current study.

Similar to the US Food and Drug Administration-approved drug idelalisib,²⁶  TGR-1202 selectively inhibits PI3Kδ. Interestingly, our results indicate that TGR-1202 is more effective than idelalisib in disrupting the 4E-BP1–eIF4F–c-Myc axis and inducing cell death in lymphoma cells, both as a single agent and in combination with proteasome inhibitors. In agreement with these preclinical data, TGR-1202 has demonstrated clinical activity in DLBCL, whereas other PI3Kδ inhibitors failed.³⁵  TGR-1202 possesses the unique capability to inhibit CK1ε (Figure 6), which distinguishes it from idelalisib. Collectively, these results support the hypothesis that cotargeting multiple regulators of 4E-BP1, including the PI3K-AKT-mTOR pathway and CK1ε (as discussed in the following paragraphs), is required for optimal suppression of translation initiation.

Previous studies in breast cancer cell lines and mouse xenografts have demonstrated that CK1ε plays an important role in promoting cancer cell proliferation by regulating phosphorylation of 4E-BP1 and initiation of mRNA translation.¹⁸  However, no CK1ε inhibitor is currently available for clinical study. The selective CK1ε inhibitor PF4800567 was very potent in the cell-free kinase assay, with an EC₅₀ of 7.4 nM (Figure 6E). Surprisingly, very high concentrations of PF4800567, in the range of 25 to 50 μM, were required to inhibit phosphorylation of 4E-BP1 (Figure 7). The reason for such discrepancy is not understood. Interestingly, autophosphorylation of CK1ε at its C terminus inhibits the kinase activity of CK1ε.³³  Conversely, it is possible that by potently inhibiting autophosphorylation, PF4800567 may somehow self-limit the inhibition of CK1ε.

In silico docking studies demonstrate that TGR-1202 binds the ATP pocket of CK1ε very well (Figure 6C-D). However, we do not yet have insights into how TGR-1202 interacts with CK1ε outside of the ATP pocket, which may be relevant to the relatively low potency of TGR-1202 with an EC₅₀ of 6.0 μM in the cell-free kinase assay. Nevertheless, the drug TGR-1202 effectively inhibited autophosphorylation of CK1ε and phosphorylation of 4E-BP1 at 15 μM in lymphoma cells (Figures 6 and 7). We speculate that the 2 structural modules of TGR-1202, 1 for targeting PI3Kδ and 1 for CK1ε, may be actually advantageous for blocking the kinase domain of CK1ε. When administered at 1200 mg once daily in the phase 1 clinical study (#NCT01767766), TGR-1202 is very well tolerated and produced a maximal plasma concentration (Cmax) of 7700 ng/mL and steady-state concentration of 5200 ng/mL,³⁷  corresponding to 13.5 and 9.1 μM. These results suggest that TGR-1202 is a first-in-class dual PI3Kδ/CK1ε inhibitor that may be adequate for targeting CK1ε in patients, especially in combination with carfilzomib.

Our results have a few clinical ramifications. The structural insights gained from TGR-1202 and CUX-03173 using in silico docking and from future crystallography studies may allow for further optimization of TGR-1202 and development of next-generation CK1ε inhibitors for cancer treatment. The combination of TGR-1202 and carfilzomib may represent a promising treatment of select patients with lymphoma that is characterized by c-Myc overexpression due to deregulated translation of c-Myc. A phase 1/2 study (NCT02867618) evaluating this regimen has been approved by the institutional review board and is open to accrual. As TGR-1202 is not associated with frequent colitis and pneumonitis,³⁸  which have been reported for idelalisib,³⁵  TGR-1202 will likely be well tolerated in combination with carfilzomib. Correlative studies of CK1ε, mTOR, 4E-BP1, and c-Myc in tumor samples before and after treatment are expected to provide insights into prognostic and predictive biomarkers for the treatment. Finally, if the combination of TGR-1202 and carfilzomib or TGR-1202 as a single agent is able to effectively turn off c-Myc, potentially, these treatments can be administered briefly and immediately before standard chemotherapy regimens as a strategy to enhance the cure potential and response rates of chemotherapy.

The authors thank Jennifer Amengual and Ahmed Sawas for critical reviewing, and Marc Brown for assistance with the computed tomography scan images. The authors thank TG Therapeutics for supplying TGR-1202.

Some experiments in this publication were performed at the Columbia Center for Translational Immunology Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056. Additional funding was provided by the Lymphoma Research Fund (C.D. and O.A.O.), TG Therapeutics (C.D.), the Columbia University Medical Center Precision Medicine Pilot Award (C.D.), and the Clinical Trial Office Pilot Award (C.D.).

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Contribution: C.D., M.R.L., L.S., X.O.J.S., M.A.M., S. Li, J.V., Y.H., X.X., and R.B.R. designed and performed the experiments and interpreted the results; S.-X.D., N.P.T., C.K., S. Lentzsch, D.A.F., B.H., D.W.L., and O.A.O. designed the experiments and provided advice; C.D. wrote the manuscript; and D.A.F., D.W.L., and O.A.O. edited the manuscript.

Conflict-of-interest disclosure: C.D. has received research funding from TG Therapeutics. The remaining authors declare no competing financial interests.

The current affiliation for M.R.L. is Division of Medical Sciences, Harvard Medical School, Boston, MA.

Correspondence: Changchun Deng, Center for Lymphoid Malignancies, Columbia University Medical Center, 51 West 51st St, 2nd Floor, New York, NY 10019; e-mail: cd2448@columbia.edu.