Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin

Simple Summary The tumor suppressor p53 is frequently mutated in human cancer. Accumulation of missense mutant p53 (mutp53) in tumors is crucial for malignant progression, and cancers are often addicted to oncogenic mutp53. However, strategies to deplete mutp53 have not yet been established. Recent studies have shown that misfolded or conformational mutp53 is stabilized by DNAJA1, a member of HSP40, also known as J-domain proteins (JDPs). However, no selective DNAJA1 inhibitor is clinically available. Through a molecular docking study, we identified a potential DNAJA1 inhibitor, called PLTFBH, derived from the natural compound plumbagin, as a compound that bound to and reduced protein levels of DNAJA1 and several other HSP40/JDPs. PLTFBH reduced the levels of conformational mutp53 and inhibited cancer cell migration in a manner dependent on DNAJA1 and mutp53. Abstract Accumulation of missense mutant p53 (mutp53) in cancers promotes malignant progression. DNAJA1, a member of HSP40 (also known as J-domain proteins: JDPs), is shown to prevent misfolded or conformational mutp53 from proteasomal degradation. Given frequent addiction of cancers to oncogenic mutp53, depleting mutp53 by DNAJA1 inhibition is a promising approach for cancer therapy. However, there is no clinically available inhibitor for DNAJA1. Our in silico molecular docking study with a natural compound-derived small molecule library identified a plumbagin derivative, PLIHZ (plumbagin–isoniazid analog), as a potential compound binding to the J domain of DNAJA1. PLIHZ efficiently reduced the levels of DNAJA1 and several conformational mutp53 with minimal impact on DNA contact mutp53 and wild-type p53 (wtp53). An analog, called PLTFBH, which showed a similar activity to PLIHZ in reducing DNAJA1 and mutp53 levels, inhibited migration of cancer cells specifically carrying conformational mutp53, but not DNA contact mutp53, p53 null, and wtp53, which was attenuated by depletion of DNAJA1 or mutp53. Moreover, PLTFBH reduced levels of multiple other HSP40/JDPs with tyrosine 7 (Y7) and/or tyrosine 8 (Y8) but failed to deplete DNAJA1 mutants with alanine substitution of these amino acids. Our study suggests PLTFBH as a potential inhibitor for multiple HSP40/JDPs.


Introduction
Discovery of novel therapeutic agents targeting cancer-specific events is a promising avenue for cancer treatment regimens [1]. The tumor suppressor p53 (p53) is the most frequently mutated gene in human cancers [2,3]. Wild-type p53 (wtp53) functions as a transcription factor that inhibits tumor development by transactivating numerous downstream target genes involved in cell cycle arrest and cell death [4]. Most mutations in
2.5. Generation of DNAJA1 and p53 Knockout or Knockdown Cell Lines DNAJA1or p53-knockout HN31 cells were generated according to the protocol described previously [20]. HN31 cells were infected with scramble control, DNAJA1 sgRNA-, or p53 sgRNA-encoding lentivectors, followed by infection with the GFP-Cas9-encoding adenoviral vector. Cells were selected by G418 or puromycin, followed by single colonization. Deletions of DNAJA1 or p53 genes were confirmed by Western blotting and then verified by DNA sequencing. Knockdown of DNAJA1 and p53 was performed using their specific short hairpin RNAs (shRNAs) as previously described [15].

Western Blotting
Cells were lysed using CelLytic M buffer (Sigma-Aldrich, Inc.) containing protease inhibitors (Thermo Fisher Scientific). Equal amounts of cell lysate were separated on a tris-glycine gel (Bio-Rad Laboratories, Hercules, CA, USA), transferred to polyvinylidene fluoride (PVDF) membranes (Amersham™ Hybond ® P 0.45 µm, Cytiva, Global Life Sciences Solutions LLC, Marlborough, MA, USA), and blotted with designated primary antibodies for each protein of interest, followed by appropriate fluorescence-tagged secondary antibodies. The blots were analyzed with the Li-Cor Odyssey infrared imaging system (Li-Cor) or the Sapphire Biomolecular Imager (Azure Biosystems Inc., Dublin, CA, USA). Full images of the Western blots with molecular weight markers, as well as the densitometry intensity ratio of each band, are presented in Supporting information-full images of Western blots.

Immunofluorescence
Cells were seeded onto Lab Tek II Chamber Slides coated with poly-D-lysine mimic (154941, Thermo Fisher Scientific). After treatment with DMSO or compounds, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. Then, cells were permeabilized with PBS containing 0.3% Triton X-100 (PBS-T) for 20 min. After blocking in 1% BSA in PBS-T for 1 h, cells were incubated with the designated primary antibodies, followed by fluorescence-tagged corresponding secondary antibodies. Samples were mounted using the ProLong Gold Antifade Reagent with DAPI (ThermoFisher). Images were analyzed using BZ-X800 Keyence All-in-One fluorescent microscope (KEYENCE CORPORATION, Itasca, IL, USA).

F-Actin Staining
Cells were seeded onto 24-well plates containing poly-L-lysine-coated 12 mm coverslips purchased from Corning (354085, Glendale, AZ, USA). Cells were treated with DMSO or PLTFBH for 16 or 24 h, fixed in 4% paraformaldehyde for 20 min, permeabilized with PBS-T for 20 min, and blocked in 1% BSA in PBS-T for 1 h. Then, the cells were incubated with phalloidin stain for 2 h, with gentle shaking, at room temperature. Quantification of filopodia formation at the circumference of cells was performed as previously described [20].

Transwell Migration Assay
Cells were pre-incubated with DMSO or PLTFBH for 12 h. The cells were then split, and viable cells were counted by trypan-blue staining. Viable cells (1-4 × 10 4 ) suspended in DMEM with 0.5% FBS containing either DMSO or PLTFBH were placed in the upper permeable cell culture inserts (24-well plate, 8.0 µm pore size, CELLTREAT Scientific Products, Pepperell, MA, USA). The bottom chamber containing DMEM with 10% FBS was used as a chemoattractant. After 12 h, migrating cells on the bottom of the membrane were fixed, permeabilized, and stained with Diff-Quik Set (Dade Behring, Deerfield, IL, USA). Migrating cells were viewed using the EVOS M5000 microscope (Thermo Fisher Scientific). The numbers of migrating cells in the entire field were counted, and the percentage of the migrating cells was calculated compared to the control group.

Cellular Thermal Shift Assay (CETSA)
Cells treated with 80 µM of DMSO or compounds for 4 h were harvested in PBS and aliquoted into thin-walled PCR tubes (Thermo Fisher Scientific), followed by incubation at different temperatures, from 40 • C to 58 • C, for 3 min. After heating, cells were incubated at room temperature for 3 min and then subjected to 3 cycles of freeze-thaw to extract proteins. Following centrifugation to precipitate insoluble aggregated proteins, the supernatants, which contained compound-bound target proteins resistant to heat-induced denaturation, were used for Western blotting for different HSP40/JDPs.

Rac1/Cdc42 Pull-Down Activation Assay
The Rac1/Cdc42 pull-down activation assay kit was purchased from (PAK02, Cytoskeleton, Inc., Denver, CO, USA). Cells treated with DMSO or PLTFBH at~1/2 IC 50 (24 h) for 16 h were lysed in the manufacturer's lysis buffer containing protease inhibitors. Cell lysates were centrifuged at 4 • C for 10 min, and the supernatant was incubated with the PAK-PBD beads at 4 • C for 2 h with rotation. After pelleting and washing the beads three times in ice-cold PBS, the beads were resuspended in SDS lysis buffer for Western blotting to detect active Rac1 and Cdc42.

Statistical Analysis
The differences between samples were analyzed by two-tailed Student's t-test or twoway Analysis of Variance (ANOVA) using the GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA). Data from at least three biological replicates were expressed as mean ± SEM, and the differences were considered statistically significant by p < 0.05.

Identification of a Compound That Binds DNAJA1 to Specifically Reduce Conformational mutp53
Currently, no specific DNAJA1 inhibitors are commercially and clinically available. To identify a potential inhibitor for DNAJA1, we conducted an in silico docking study using a natural compound library against the J domain of DNAJA1 (PDB: 2LO1) and identified a plumbagin-derived PLIHZ as a compound that could bind to DNAJA1. The study

Identification of a Compound That Binds DNAJA1 to Specifically Reduce Conformational mutp53
Currently, no specific DNAJA1 inhibitors are commercially and clinically available. To identify a potential inhibitor for DNAJA1, we conducted an in silico docking study using a natural compound library against the J domain of DNAJA1 (PDB: 2LO1) and identified a plumbagin-derived PLIHZ as a compound that could bind to DNAJA1. The study also identified tyrosine 7 (Y7) as a critical amino acid for the PLIHZ-DNAJA1 interaction with binding energy of −6.6 kcal/mol at 2.6 Å distance (Figure 2A). PLIHZ was previously identified as a compound derived from a phytochemical plumbagin [22,25]. Although plumbagin regulates Akt signaling and p53 activity to show anti-tumor effects in multiple types of cancer [22,[26][27][28], the biological effects of its derivative PLIHZ and the underlying mechanisms remain unclear. To validate the DNAJA1-PLIHZ binding within cells, we performed a cellular thermal shift assay (CETSA), in which a compound-bound target protein becomes resistant to heat-induced protein denaturation and aggregation, causing the protein-compound complex to be retained in the soluble fraction after centrifugation of cell lysates [29][30][31]. In both CAL33 and HN31 cells, DNAJA1 protein levels were significantly increased between 46 °C and 55 °C in the supernatants of cells treated with PLIHZ as compared to the vehicle control ( Figure 2B and Supplementary Figure S2A). These results suggest increased thermal stability of DNAJA1 protein by PLIHZ, thereby demonstrating physical binding between PLIHZ and DNAJA1 in cells.  To validate the DNAJA1-PLIHZ binding within cells, we performed a cellular thermal shift assay (CETSA), in which a compound-bound target protein becomes resistant to heatinduced protein denaturation and aggregation, causing the protein-compound complex to be retained in the soluble fraction after centrifugation of cell lysates [29][30][31]. In both CAL33 and HN31 cells, DNAJA1 protein levels were significantly increased between 46 • C and 55 • C in the supernatants of cells treated with PLIHZ as compared to the vehicle control ( Figure 2B and Supplementary Figure S2A). These results suggest increased thermal stability of DNAJA1 protein by PLIHZ, thereby demonstrating physical binding between PLIHZ and DNAJA1 in cells.
Next, we determined the 24h-IC 50 values of PLIHZ in multiple cancer and non-tumor cell lines with different p53 status. Cells harboring conformational mutp53 exhibited lower 24h-IC 50 values for PLIHZ, compared to cells with DNA contact mutp53, p53 null, or wtp53, regardless of cancer cells or non-tumor cells (Supplementary Figure S2B). Hereafter, we used approximately half (1/2) of the IC 50 values for experiments unless otherwise noted. We then treated cancer cells with different p53 status, including HN31, MDA-MB-231, U2OS, and H1299, with~1/2 24h-IC 50 of PLIHZ. We found that PLIHZ reduced protein levels of conformational mutp53 in HN31 cells with little effect on the p53 levels in MDA-MB-231, U2OS, and H1299 cells by Western blotting ( Figure 2C). Intriguingly, PLIHZ reduced the DNAJA1 levels in all cell lines examined. Essentially the same results were observed using immunofluorescence studies ( Figure 2D and Supplementary Figure S2C). These findings suggest that PLIHZ likely binds to DNAJA1 in all cells to reduce the protein level of DNAJA1; however, the reduction in DNAJA1 protein only alters the level of conformational mutp53, consistent with the results in Figure 1.

PLTFBH, An Analog of PLIHZ, Specifically Reduces Conformational mutp53 Levels Similar to PLIHZ
To improve the biological and chemical properties of PLIHZ, we synthesized four PLIHZ analogs, called PLFBH, PLTFBH, PLFUH, and PLOCT ( Figure 3A). We compared the efficacy of PLIHZ and these analogs on reducing the protein level of conformational mutp53 by Western blotting. All analogs, except PLOCT, exhibited comparable potency in reducing the protein levels of DNAJA1 and conformational mutp53 in HN31 cells, as well as CAL33 and KHOS/NP cells ( Figure 3B and Supplementary Figure S3A). Importantly, none of these analogs reduced the protein levels of wtp53 and DNA contact mutp53 (R248L, R273H) in SJSA1, HN30, FaDu, and HT29 cells (Supplementary Figure S3A). When we determined 72h-IC 50 for these PLIHZ analogs, all analogs, except PLOCT, showed similar cytotoxic activities in cancer cell lines harboring conformational mutp53 including HN31, KHOS/NP, and CAL33 (Supplementary Figure S3B). Although PLIHZ, PLFBH, PLTFBH, and PLFUH showed comparable conformational mutp53-depleting and cytotoxic activities to cancer cells, PLTFBH consistently reduced the levels of multiple conformational mutp53 better than other analogs with minimal impact on wtp53 and DNA contact mutp53 ( Figure 3B and Supplementary Figure S3A). Hence, hereafter, we focused on the PLTFBH compound.
Next, we determined 24h-IC 50 values of PLTFBH in multiple cancer and non-tumor cell lines with different p53 status. Similar to PLIHZ, cells expressing conformational mutp53 showed relatively lower 24h-IC 50 values for PLTFBH, compared to those expressing DNA contact mutp53, p53 null, or wtp53 (Supplementary Figure S3C). We then examined the effects of PLTFBH at 1/2 of 24h-IC 50 on the levels of DNAJA1 and p53 in cancer cell lines with different p53 status. Similar to PLIHZ, PLTFBH reduced the levels of only conformational mutp53, although it decreased DNAJA1 levels in all cell lines examined by Western blotting ( Figure 3C) and immunofluorescence ( Figure 3D). We also confirmed the intracellular interaction of PLTFBH with DNAJA1 in CAL33 and HN31 cells by CETSA ( Figure 3E and Supplementary Figure S3D).
To further explore dependency of the cytotoxic effects of PLTFBH on DNAJA1 and mutp53, we genetically deleted DNAJA1 or mutp53 in HN31 cells by the CRISPR-Cas9 strategy ( Figure 3F). These HN31 sub-cell lines were treated with different concentrations of PLTFBH for 72 h, followed by MTT assays ( Figure 3G). Although deletion of DNAJA1 or mutp53 made HN31 cells slightly more resistant to PLTFBH, the differences in the 72h-IC 50 values were not statistically significant. These data suggest that the cytotoxic effect of PLTFBH is not entirely dependent on DNAJA1 and mutp53, and PLTFBH may have other biological targets in addition to DNAJA1.

PLTFBH Inhibits Migratory Potential of Cancer Cells in a Manner Dependent on DNAJA1 and Conformational mutp53
One of the major mutp53 GOF activities is to enhance cancer cell migration and metastasis [9,10,32,33]. Moreover, depletion of DNAJA1 results in reduced filopodia formation and migratory potential of cancer cells expressing conformational mutp53 [15,20]. To examine the effect of PLTFBH on DNAJA1-and conformational mutp53-dependent migration, we measured the migratory potential of cells with different p53 status, following PLTFBH treatment. As expected, PLTFBH significantly inhibited the migration of cancer cells harboring conformational mutp53 (HN31, KHOS/NP). In contrast, the migratory potential of cells harboring DNA contact mutp53 (MDA-MB-231, Panc-1), wtp53 (U2OS, SJSA1, Het-1a), and p53 null (H1299, SAS) was not altered by PLTFBH ( Figure 4A). We also examined the effects of PLTFBH on filopodia formation of cancer cells with different p53 status. Consistently, PLTFBH inhibited filopodia formation in HN31 and CAL33 cells harboring conformational mutp53, whereas it had minimal impact on filopodia formation in cancer cells with DNA contact mutp53, wtp53, and p53 null ( Figure 4B and Supplementary Figure S4A). Moreover, knockdown of either DNAJA1 or conformational mutp53 abrogated the PLTFBH-mediated inhibition of migration of HN31 and KHOS/NP cells ( Figure 4C and Supplementary Figure S4B).    The signaling involved in filopodia formation and migration is regulated by the activity of Cdc42 and Rac1 [20,34,35]. Moreover, knockdown of DNAJA1 inhibits the activity of Cdc42 and Rac1 in HNSCC cells [20]. Hence, we examined the effects of PLTFBH on the Cdc42/Rac1 activities in HN31 and MDA-MB-231 cells. In agreement with the results of inhibited filopodia formation and migration by PLTFBH, PLTFBH reduced the active forms of Cdc42 and Rac1 in HN31 cells with a conformational mutp53, whereas it had minimal effects on the Cdc42 and Rac1 activities in MDA-MB-231 cells carrying a DNA contact mutp53 ( Figure 4D and Supplementary Figure S4C). Together, these results strongly suggest that PLTFBH inhibits migratory potential of cancer cells predominantly in a manner dependent on DNAJA1 and mutp53, demonstrating specificity of PLTFBH for these targets.
We also excluded the possibility that DNAJA1 depletion could lead to reduced protein levels of some of HSP40/JDPs. To address this concern, we examined the effects of DNAJA1 knockout on the protein levels of several PLTFBH-responding HSP40/JDPs (DNAJB1, DNAJB12, DNAJC3, DNAJC7), as well as a non-responding DNAJC6, using control and DNAJA1-knockout HN31 cells. There was no change in the protein levels of these HSP40/JDPs by DNAJA1 knockout, confirming that reduced protein levels of some HSP40/JDPs were not due to DNAJA1 depletion (Supplementary Figure S5B). DNAJC8, DNAJC10, DNAJC15, and DNAJC20, in HN31 cells. Besides DNAJA1, PLTFBH decreased the level of several other HSP40/JDPs to variable extents ( Figure 5A). These results were further confirmed by immunofluorescence studies (Supplementary Figure  S5A). We arbitrary classified their responses into three groups of good (DNAJA1, DNAJA2, DNAJA3, DNAJB1, DNAJB12, and DNAJC3), moderate (DNAJA4, DNAJB2, DNAJB6, DNAJC2, DNAJC7, DNAJC10, and DNAJC20), and little or no (DNAJC6, DNAJC8, and DNAJC15) ( Figure 5B). Our docking analyses identified Y7 in the J domain of DNAJA1 as a putative amino acid critical for the PLIHZ-DNAJA1 binding. Intriguingly, HSP40/JDPs lacking both Y7 and its neighboring amino acid Y8 (DNAJC6, DNAJC8, DNAJC15) failed to respond to PLTFBH, while PLTFBH reduced the protein levels of HSP40/JDPs with either or both of these amino acids to variable extents ( Figure 5C). We next performed CETSA to determine whether PLTFBH could bind with DNAJA4 (moderate responder with both Y7 and Y8) and/or DNAJC6 (little or no responder lacking both Y7 and Y8) with PLTFBH using CAL33 cells. Consistent with the results above, PLTFBH successfully bound to DNAJA4; however, it failed to bind to DNAJC6 ( Figure 5D). These results may suggest that the protein structure near residues Y7 and Y8 is crucial for PLTFBH's binding to the J domain of HSP40/JDPs, resulting in depletion of the target proteins.
3.6. Mutations at Y7 and Y8 Residues in DNAJA1 Abrogate the Ability of PLTFBH to Deplete DNAJA1 and Conformational mutp53 To further delineate the significance of Y7 and Y8 in DNAJA1 for the action of PLTFBH, we re-expressed wild-type DNAJA1 (wt), a mutant DNAJA1 with Y7 substituted to alanine (Y7A), or a mutant DNAJA1 with Y8 substituted to alanine (Y8A) in DNAJA1-knockout HN31 cells, followed by Western blotting ( Figure 6A) and immunofluorescence ( Figure 6B). In the absence of DNAJA1, PLTFBH showed little effect on the protein level of endogenous conformational mutp53 (p53 C176F ). Re-introduction of wt or mutant DNAJA1 (Y7A, Y8A) resulted in rescue of mutp53 protein levels. PLTFBH treatment successfully reduced exogenous wild-type DNAJA1 (wt) and endogenous mutp53 protein levels. However, it failed to reduce exogenous Y7A and Y8A mutant DNAJA1. Accordingly, mutp53 levels in these mutant DNAJA1-expressing cells were unchanged ( Figure 6A,B). We additionally confirmed reduction in endogenous DNAJB6 proteins by the PLTFBH treatment in these HN31 sub-cell lines (Supplementary Figure S6A).  We furthermore assessed the effects of mutant DNAJA1 (Y7A, Y8A), which rescued the endogenous mutp53 protein levels but did not respond to PLTFBH, on filopodia formation using these HN31 sub-cell lines ( Figure 6C). Exogenous expression of wt or mutant DNAJA1 restored filopodia formation, consistent with the restored endogenous mutp53 levels. As expected, PLTFBH inhibited the filopodia formation induced by wt DNAJA1; however, it had minimal impact on the filopodia formation induced by Y7A and Y8A DNAJA1 mutants. Taken together, these results furthermore confirmed critical roles of Y7 and Y8 of DNAJA1 in PLTFBH-mediated depletion of DNAJA1 and conformational mutp53, leading to suppression of filopodia formation and migration.

Discussion
Our in silico docking-based study has identified a plumbagin-derivative PLIHZ and its analog PLTFBH as compounds that bind to the J domain of DNAJA1 and induce depletion of multiple conformational mutp53. PLIHZ is a synthetic naphthoquinone derived from the combination of the natural phytochemical agent, plumbagin, and the anti-tuberculosis agent, isonicotinic hydrazid (INH) [25]. Plumbagin is isolated from the roots of the medicinal plant Plumbago zeylanica and has been suggested as an anti-cancer, anti-inflammatory, and cytotoxic agent [26,37]. Plumbagin shows its anti-tumor effects through induction of cell cycle arrest, apoptosis, and autophagy, as well as inhibition of EMT by inhibition of Akt signaling, activation of wtp53, inhibition of NF-kB activity, and other unknown mechanisms, in multiple types of cancer [22,[26][27][28]. However, the effects of the plumbagin analog PLIHZ on cellular signaling and cancer progression have not been tested [22,25].
We confirmed the intracellular binding of PLIHZ and its derivative PLTFBH to DNAJA1 by CETSA and their effects on reducing protein levels of conformational mutp53, but not wtp53 and DNA contact mutp53. Intriguingly, both PLIHZ and PLTFBH reduced the protein levels of DNAJA1 as well, although the underlying mechanism remains to be determined. Unfortunately, these compounds showed non-specific inhibition of viable cell proliferation, based on their cytotoxic effects in DNAJA1 and/or mutp53 knockout cells; however, PLTFBH showed specific inhibition of filopodia formation and migration of cancer cells expressing conformational mutp53 with minimal impact on cells with wtp53, p53 null, and DNA contact mutp53. Moreover, PLTFBH had minimal impact on the migration of cells lacking DNAJA1 and mutp53, demonstrating the on-target effects. Thus, to the best of our knowledge, this is the first study showing the efficient depletion of mutp53 and subsequent inhibition of cancer cell migration through inhibition or depletion of DNAJA1 by a natural product-derived compound.
Previously, Moses et al. [19] reported a potential HSP40/JDP inhibitor, chalcone C86, that induced degradation of androgen receptor (AR) and its variant ARv, similar to HSP70 inhibitors (JG98, JG231), by binding to the J domain of multiple HSP40/JDP members. Intriguingly, C86 does not alter the levels of HSP40/JDPs, unlike the case of PLIHZ or PLTFBH. Since their study does not use HSP40/JDPs knockout/knockdown cells, it remains unclear if the observed inhibition of HSP40/JDPs by C86 is the direct cause of depletion of AR and ARv and which HSP40/JDPs play roles in this activity. Additionally, side-by-side comparison studies of C86 and PLTFBH for their efficacy to reduce mutp53 levels would be important as a future study.
One major caveat associated with PLTFBH treatment is the induction of some cytotoxicity in cancer cells lacking DNAJA1 or mutp53. This observation may suggest that other HSP40/JDP proteins whose activities or levels are reduced by PLTFBH could regulate cancer cell proliferation or survival. Hence, DNAJA1-and mutp53-independent cytotoxic effects of PLTFBH could be explained by depletion of other HSP40/JDPs than DNAJA1. Further studies are required to clarify which other HSP40/JDPs could contribute to cytotoxic effects of PLTFBH. More importantly, it needs to be carefully determined whether PLIHZ analogs can be used for anti-cancer therapies, since they could impact the levels and activities of client proteins of other HSP40/JDPs.
Our group has recently published that DNAJA1 protein specifically binds to misfolded or conformational mutp53 but not wtp53 or DNA contact mutp53 with relatively intact p53 protein structure [20]. The biological effects of DNAJA1 in cancer cells are largely dependent on the presence of conformational mutp53; DNAJA1 promotes filopodia formation and cancer cell migration by binding to and stabilizing misfolded or conformational mutp53. These observations are in line with our finding that depletion or inhibition of DNAJA1 by PLTFBH reduces filopodia formation and migration of cancer cells specifically expressing conformational mutp53. It should also be noted that PLTFBH shows the minimal impact on the viability of non-tumor cell lines, which could suggest a therapeutic range. The pharmacological properties of PLTFBH have not yet been characterized. Following improvement of the efficacy and specificity of PLTFBH analogs to DNAJA1 and evaluation of their pharmacological properties, it is crucial to test their in vivo effects on tumor progression, as well as toxicity and safety, using pre-clinical studies. This would accelerate the development of the future DNAJA1-mutp53-targeted therapies with minimal side effects.

Conclusions
We identify PLTFBH, derived from the natural compound plumbagin, as a compound that binds to the J domain of DNAJA1, through a molecular docking study. This compound binds to and reduces protein levels of DNAJA1 as well as conformational mutp53, leading to inhibited cancer cell migration. This work highlights DNAJA1 as a therapeutic target in cancers carrying conformational mutp53, as well as the use of PLTFBH as an inhibitor of DNAJA1 and certain members of HSP40/JDPs.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cancers14174187/s1, Figure S1: Knockdown of DNAJA1 specifically reduces protein levels of conformational mutp53, but not DNA contact mutp53 and wtp53. Figure S2: Binding of PLIHZ to DNAJA1 and the effects on cell viability and protein levels of DNAJA1 and p53 in multiple human cell lines with different p53 status. Figure S3: Effects of PLIHZ analogs on mutp53 levels, cell viability, and binding to DNAJA1. Figure S4: PLTFBH inhibits migratory potential of cancer cells in a manner dependent on DNAJA1 and conformational mutp53. Figure S5: PLTFBH selectively decreases protein levels of certain members of HSP40/JDPs. Figure S6: Mutations at Y7 and Y8 residues in DNAJA1 abrogates the ability of PLTFBH to deplete DNAJA1 and conformational mutp53.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.