The clinical, pathological and molecular features of 498 untreated diffuse large B-cell lymphoma (DLBCL) were previously described by us (15–17). Their gene expression data from microarray analyses are publicly available (GSE31312). Overall survival (OS) was calculated from the time of diagnosis to the last follow-up or death due to any cause. Progression-free survival (PFS) was calculated from the time of diagnosis to disease progression (relapse or death) or last follow-up.

Seventeen de-identified frozen biopsies from patients with untreated DLBCL of the germinal center B cell (GCB-DLBCL) or activated B cell (ABC-DLBCL) origin were obtained from an archive maintained by the Department of Pathology at the University of Texas Health Science Center at San Antonio (UTHSCSA). The clinical, pathological and molecular features of these tumors were previously described (18) and are listed in Table 1 . Three de-identified surgically removed tonsils were obtained from the University Health System hospital with the donors’ consent. All studies were approved by the UTHSCSA institutional review board.

Human naïve IgM⁺IgD⁺ B cells were purified by negative selection from peripheral blood mononuclear cells (PBMCs) obtained from healthy subject buffy coat (South Texas Blood & Tissue Center, San Antonio) using the EasySep Human Naïve B Cell Enrichment Kit (STEMCELL Technologies, cat# 19254). For activation, human naïve B cells (10⁶ cells/ml) were stimulated with CpG ODN2395 (2.5 mg/ml; invivogen), plus recombinant human IL-21 (50 ng/ml; BioLegend) for 48 hours, as described (19).

B lymphoma cell lines included those established from Burkitt lymphoma (Daudi and CL-01), GCB-DLBCL (OCI-Ly7) or ABC-DLBCL (OCI-Ly10 and U2932). Other cell lines included Nalm-6 B leukemia, Jurkat T leukemia, SK-MEL-28 and UACC-62 melanoma, and U251 and U343 glioblastoma. All cell lines were cultured in RPMI 1640 medium supplemented with FBS (10% v/v, ThermoFisher) and penicillin-streptomycin/amphotericin B (1% v/v) (RPMI-FBS) at 37°C with 5% CO₂.

Daudi cell viability was checked (>95% live cells) before subcutaneous injection of 3×10⁶ cells into Foxn1^nu/nu nude mice (JAX, Stock# 002019). Tumor sizes were measured every week. Length × width × width × ½ is used to calculate the size of the tumor. Mice with a tumor over 2500 mm³ in size were euthanized after one more measurement. Mice were euthanized by gradual displacement of room air with CO₂ at approximately 30% of the chamber volume per minute until respiration ceased. Death was confirmed by cervical dislocation. The protocol was in accordance with the rules and regulations of the Institutional Animal Care and Use Committee (IACUC) of UTHSCSA.

Cell pellets (5×10⁶ cells) were washed twice with cold PBS and lysed by sonication in PBS plus 1% Tween-20. After the protein concentration in cell lysates was determined, 20 μg lysates from each sample were analyzed by SDS-PAGE and Western blotting using anti-RAB7 antibody (Abcam; cat# ab137029) and anti-β-actin (Cell Signaling Technology, CST; cat# 4970).

For flow cytometry analysis, cell pellets (1×10⁶ cells) were washed once in cold Hank’s Buffered Salt Solution (HBSS) with 1% BSA and stained with fixable viability dye (FVS; BioLegend) and fluorophore-labeled mAbs to CD19 (BD biosciences; cat# 553785) and IgD (BD; cat# 562518). The cells were fixed and stained intracellularly with fluorophore-labeled mAb to RAB7 (Abcam; cat# ab198337), total RELA (CST; cat# 9609) and phosphorylated RELA (CST; cat# 4886), following the instructions of the BD intracellular staining kit (cat# 554714). Dead (FVS⁺) cells were excluded from the analysis.

For in vivo treatment, CID1067700 (Glixx Laboratories) dissolved in DMSO (stock concentration 40 mM, 16 mg/ml) was diluted to the final volume of 50 μl and injected intraperitoneally (i. p.) into Foxn1^nu/nu mice daily from day 7 to day 12 at the dose of 16 mg/kg body weight. Mice injected i. p. with the vehicle DMSO (nil; 50 μl) showed no difference in tumor progression compared to those without any injection (data not shown). For in vitro treatment, the CID1067700 stock solution was diluted in DMSO to make 100× working solutions and then added to cell cultures to indicated final concentrations. To treat cells with methyl-β-cyclodextrin (MβCD, Sigma Aldrich), MβCD was freshly dissolved in RPMI-FBS to make a stock solution of 16 mM (21 mg/ml) and then further diluted with RPMI-FBS to make 4× working solutions before adding to cell cultures. To treat cells with FX1 (Selleck Chemicals), 10 mM solution in DMSO was diluted with RPMI-FBS to make 4× working solutions before adding to cell cultures.

RNA was extracted from biopsies or B cells (2×10⁶) using the RNeasy Mini Kit (Qiagen). First-strand complementary DNA was synthesized from equal amounts of total RNA (4 μg) using the SuperScript III System (Invitrogen) and analyzed by the SYBR Green dye incorporation. The ΔΔCt method was used to determine transcript levels after normalizing to that of ACTB (encoding β-actin). qPCR primers used are:

Cells were seeded at 0.5×10⁶ cells/ml in RPMI-FBS and cultured for 24 hours and then re-seeded at 0.5×10⁶ cells/ml in 100 µl RPMI-FBS in the presence of drug or vehicle in a flat-bottom 96-well plate. After 24 or 48 hours, 20 µl MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]; Promega; cat# G5421) was added. The plate was read 4 hours later at the 490 nm and 670 nm wavelengths. After subtracting the reading at 670 nm, the reading at 490 nm was used to measure cell growth after further subtraction of the background levels from RPMI-FBS medium only.

B cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (ThermoFisher) following the manufacturer’s instructions and then cultured for 96 h. Cells were analyzed by flow cytometry for the CFSE intensity, which was reduced by half after completion of each cell division, when CFSE-labeled cell constituents were equally distributed between daughter cells. The FlowJo^® software was used to determine the number of B cell divisions that B cells had completed. The viability was analyzed by staining cells with 7-AAD followed by FACS to quantify 7-AAD^– live cells.

DLBCL survival rate difference between two groups were compared using the Kaplan-Meier method and log-rank test in GraphPad Prism 10 software. Student t test or ANOVA was performed to compare group mean. The mixed linear effect model test was used to analyze the in vivo tumor growth and dose-dependent cell survival in combined treatments of CID1067700 with FX1 or MβCD.

As RAB7 is expressed by murine naïve B cells and further upregulated in activated B cells, we examined its expression in human B cells. RAB7 was expressed at higher levels in tonsil CD19⁺IgD⁺ B cells (primarily naïve with some activated IgD⁺ cells) and CD19⁺IgD^– B cells (activated/switching B cells and switched memory B cells) than in CD19^– non-B cells ( Figures 1A, B ). RAB7 expression was also higher in IgD^– B cells than in IgD⁺ B cells. Both B cell subsets displayed similar levels of the RELA (p65) subunit of NF-κB to CD19^– non-B cells, but higher levels of RELA phosphorylation at Ser536, an indicator of NF-κB activation, than non-B cells ( Figures 1A, B ). They, however, showed no significant difference in RELA phosphorylation or in the ratio of phosphorylate to total RELA ( Figures 1A, B ), likely reflecting the role of NF-κB activation in both B cell development and differentiation. Overall, RAB7 was expressed in primary human B lineage cells, including activated B cells.

Prompted by heightened RAB7 expression is hyperactivated mature B cells, such as human and murine lupus B cells (11), we tested the hypothesis that RAB7 was also upregulated in mature B cell-derived lymphoma cells. We first analyzed RAB7A expression in biopsy samples from a small cohort of 17 DLBCL patients with well-defined clinical, hematological and pathological features ( Table 1 ). The transcript level RAB7A was readily detectable in all samples, as were those of BCL6, a proto-oncogene frequently overexpressed in GCB-DLBCL, and PRDM1, a tumor suppressor gene frequently downregulated in ABC-DLBCL ( Figure 2A ). Notably, the expression of RAB7A, but not BCL6 or PRDM1, correlated strongly with CD79B, a B-cell hallmark gene ( Figure 2A ), suggesting that malignant B cells, rather than tumor-infiltrating non-B cells, were the major contributors to RAB7A expression. In addition, RAB7A expression was similar in GCB-DLBCLs (n=11) and ABC-DLBCLs (n=6) at similar levels, indicating that RAB7 was broadly involved in most DLBCLs ( Figure 2B ).

To further confirm high RAB7 expression in malignant B cells, we analyzed a panel of cell lines derived from human mature B cell lymphomas, including Burkitt lymphoma, GCB-DLBCL and ABC-DLBCL. Among these, the Daudi and CL-01 Burkitt lymphoma cells as well as the OCI-Ly10 and U2932 ABC-DLBCL cells expressed RAB7 at high levels, whereas the OCI-Ly7 GCB-DLBCL cells exhibited a double-peak and relatively lower RAB7 expression pattern in flow cytometry analysis ( Figure 2C ). As comparisons, the Nalm-6 B leukemia and Jurkat T leukemia cell lines showed low RAB7 expression. The differential RAB7 expression in Daudi and Nalm-6 cells was confirmed by Western blotting ( Figure 2D ). Finally, RAB7 expression in B lymphoma cell lines was higher than that in B cells isolated from the PBMC of healthy donors and activated by TLR9 ligand CpG plus IL-21, and even higher than in naïve B cells ( Figure 2C ).

Thus, RAB7 was expressed in mature B cell-derived lymphomas irrespective the origin of the malignancies, at levels higher than those in normal B cells.

Prompted by the heightened RAB7 expression in primary DLBCLs and B lymphoma cell lines, we next tested the hypothesis that higher RAB7 expression was associated with unfavorable outcomes in DLBCL. Using mRNA microarray data (GSE31312) of a mid-sized (n=498) cohort of DLBCLs, we analyzed the correlation between the expression of selected genes prior to treatment and patient survival. Within this cohort, the RAB7A^High group exhibited significantly reduced OS (p = 0.011), with a reduction in PFS also approaching significant (p = 0.048) – PFS is an indicator of therapeutic resistance ( Figure 3A ). By comparison, high transcript levels of RAB7B, which encodes a homolog of RAB7 (50% homology), were not associated with reduced survival. The reduction in OS in the RELA^High group compared with the RELA^Low group approached significant (p = 0.05). In contrast to RAB7A, high expression of RAB5A was associated with better survival ( Figure 3B ), possibly reflecting heightened RAB5A activity that might inhibit the biogenesis of RAB7⁺ mature endosomes from RAB5A⁺ immature endosomes. High expression of RAB7L1 was also associated with better survival, possibly reflecting increased diversion of RAB7⁺ mature endosomes into the endosome-Golgi pathway, leading to reduced RAB7 activity. We also divided the cohort into four groups, RAB7A^HighRAB7B^High , RAB7A^HighRAB7B^Low , RAB7A^LowRAB7B^High and RAB7A^LowRAB7B^Low . The RAB7A^LowRAB7B^Low group displayed better OS compared to the RAB7A^HighRAB7B^Low group ( Figure 3C ). This, together with the lack of the significant difference between the RAB7A^LowRAB7B^High and RAB7A^HighRAB7B^High groups, suggested that high RAB7B levels might narrow the difference in OS caused by differential RAB7A expression, probably due to compensatory effects.

In summary, RAB7A expression was associated with unfavorable prognosis in DLBCL, suggesting that RAB7 played a role in promoting DLBCL pathogenesis and/or therapeutic resistance.

To explore whether targeting RAB7, as highly expressed in B lymphoma cell lines, could have therapeutic effects, we first treated these cells in vitro with different doses of CID1067700 and assessed viability using the MTS assay at 24 and 48 hours. At 24 hours, all five cell lines, Daudi, CL-01, OCI-Ly7, OCI-Ly10 and U2932, exhibited a dose-dependent reduction in growth, with IC50 values ranging from 53 μM to 103 μM ( Figure 4A ). At 48 hours, the IC50 values slightly decreased in all cell lines except OCI-Ly10, ranging from 62 μM to 95 μM.

To determine whether the impact of CID1067700 was specific to B cell malignancies, we treated a panel of non-B lymphoma tumor cell lines, along with Daudi and CL-01 cells, with 0, 20 or 40 μM CID1067700 and monitored the total cell numbers at 24 hours and 60 hours. CID1067700 reduced the growth of Daudi and CL-01 cells in this assay, complementing the MTS results, which showed that 20 μM and 40 μM were the lowest effective doses of CID1067700 inhibiting the growth of these cells at 48 and 24 hours, respectively ( Figure 4B ). By contrast, Nalm-6 cells were resistant to the CID1067700 treatment and Jurkat cells showed a slight reduction only at 40 μM. Minimal to no inhibition was observed in SK-MEL-28 and UACC-62 melanoma cells or in U251 and U343 glioblastoma cells, indicating that the effects of CID1067700 were specific to B cell-derived lymphomas.

Taken together, the CID1067700 treatment arrested the in vitro growth of B cell lymphoma-derived cell lines, but not other cancer lines tested here.

To determine whether the reduced growth of B lymphoma cells observed with the CID1067700 treatment was due to increased cell death, impaired proliferation, or both, we treated Daudi cells with CID1067700 or vehicle (DMSO) for 24 and 48 hours. CID1067700 treatment resulted in a significant decrease in the proportion of live cells compared with DMSO treatment, to a comparable extent at both time points ( Figure 5A ), indicating that RAB7 inhibition reduced Daudi cell viability.

To investigate whether the RAB7 activity was required for B lymphoma cell proliferation, we analyzed Daudi cell divisions upon the CID1067700 treatment using the CFSE assay. At 24 hours, CID1067700 treatment resulted in a delay in CFSE halving, indicating impaired cell proliferation ( Figure 5B , top). This was quantified by the decrease in the average cell division, from 1.5 in DMSO-treated control cells to 1.2 in CID1067700-treated cells, representing 20% reduction. At 48 hours, both CID1067700- and DMSO-treated Daudi cells exhibited further CFSE halving, with CID1067700-treated cells showing the average cell division of 2.2 compared to 3.3 in control cells ( Figure 5B , bottom), a 33% reduction, more pronounced than that at 24 hours and likely contributing to the lower IC50 at 48 hours relative to 24 hours.

Overall, RAB7 inhibition in B lymphoma cells resulted in impaired viability and proliferation, both of which likely accounted for the arrested cell growth.

We next assessed the effect of CID1067700 in a murine in vivo model of B cell lymphoma. Consistent with the low toxicity of CID1067700 previously reported in several mouse models by us (11) and others (20, 21), Foxn1^nu/nu nude mice treated with CID1067700 at a dose of 16 mg/kg body weight did not show any overt signs of diseases (data not shown). After being engrafted with Daudi cells via the subcutaneous route, mice received 6 daily intraperitoneal injections of CID1067700 or DMSO starting on day 8. CID1067700-treated mice exhibited delayed tumor progression compared with control mice, with difference becoming apparent at day 28 ( Figure 6 ), and no obvious side effects were observed during 42 days of monitoring (data not shown). These data showed that RAB7 inhibition arrested B lymphoma cell growth in vivo through a mechanism independent of T cell-mediated immunity.

We next investigated whether combination strategies could potentiate the suppression of Daudi cell growth by CID1067700. Our previous work has demonstrated that lipid rafts help anchor signaling receptors such as CD40 and TLR4 at the plasma membrane, where they initiate signal transduction. Treatment with MβCD, which disrupts these lipid rafts and associated CD40 signaling (22–24), diverts internalized receptors toward RAB7⁺ mature endosomes, where the multi-valent interactions among RAB7, CD40 and TRAF6 promote TRAF6 Lys63 polyubiquitination for heightened NF-κB activation in murine B cells (12). Accordingly, the combined treatment with MβCD and CID1067700 blocks both plasma membrane and intracellular membrane signaling pathways, leading to abrogated NF-κB activation and severely compromised B cell viability and differentiation (12). Treating Daudi B cells with 10 μM CID1067700 and 3.5 mM MβCD resulted in over 50% decrease in cell growth ( Figure 7A ). This indicated that the IC50 of CID1067700 was reduced to less than 10 μM in the presence of 3.5 mM MβCD, representing a more than eight-fold reduction compared with the IC50 of CID1067700 alone (80 μM). Combined treatment with a 1.5 mM MβCD, however, did not reduce the IC50. Notably, whereas treatment with 3.5 mM MβCD alone had no impact on Daudi cell growth, treatment with 1.5 mM MβCD alone increased the growth by 20%, similar to the increase in the NF-κB activation and CSR in murine B cells treated with MβCD alone, as previously shown (12).

FX1 is a selective BCL6 inhibitor identified through a rational design approach (25). Daudi cells treated with 80 μM or 20 μM FX1 displayed further decrease in the growth across nearly all CID1067700 doses tested ( Figure 7B ). This included 40% decrease when cells were treated with 80 μM FX1 and 10 μM CID1067700, resulting in an IC50 for CID1067700 slightly above 10 μM, which was still a significant reduction compared to the IC50 of CID1067700 alone. The FX1 treatment alone had only a marginal impact ( Figure 7B ).

In summary, inhibition of B cell lymphoma growth by the RAB7 inhibitor CID1067700 could be further enhanced by MβCD and FX1, which per se have no significant suppressive effects at the doses tested.