CaMKK2 Identifies Biologically Aggressive Chronic Lymphocytic Leukemia and Regulates Leukemic Survival and Nurse-Like Cell Support

31 Background/Objectives: Identification of prognostic biomarkers that capture biologically 32 aggressive disease remains a major need in chronic lymphocytic leukemia (CLL). Aberrant 33 calcium signaling contributes to leukemic survival; however, the clinical relevance of 34 Ca²⁺/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a calcium-responsive kinase, has 35 not been defined. This study evaluated CaMKK2 as a candidate prognostic biomarker and 36 functional regulator in CLL. Methods: CaMKK2 expression was quantified in purified CD19⁺ 37 CLL cells from a clinically annotated cohort balanced by immunoglobulin heavy chain variable 38 region (IGHV) mutation status. Associations with time-to-treatment and overall survival were 39 analyzed. Functional relevance was assessed by pharmacologic inhibition of CaMKK2 in 40 primary CLL cells using metabolic (MTS) and apoptosis (Annexin V/PI) assays. Correlations 41 between CaMKK2 expression and inhibitor sensitivity were determined. The impact of 42 CaMKK2 inhibition on nurse-like cell (NLC) differentiation and macrophage-mediated leukemic 43 support was evaluated in ex vivo culture systems. Results: Elevated CaMKK2 expression was 44 enriched in IGHV-unmutated CLL and associated with shorter time-to-treatment and inferior 45 overall survival. CaMKK2 inhibition reduced primary CLL viability in a dose-dependent manner 46 and induced apoptosis, with sensitivity correlating with CaMKK2 expression levels. Inhibition 47 also attenuated CD163⁺ macrophage polarization and impaired NLC-mediated support of 48 leukemic cells. Conclusions: CaMKK2 expression identifies biologically aggressive CLL and 49 functionally contributes to leukemic persistence. These findings position CaMKK2 as a 50 prognostically relevant biomarker with therapeutic implications, supporting further evaluation of 51 CaMKK2-targeted strategies in high-risk CLL. 52 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 3 Key words: Chronic lymphocytic leukemia; CaMKK2; Calcium signaling; IGHV-unmutated 53 CLL, Nurse-like cells; Tumor-Associated Macrophage; Tumor microenvironment; CAR-T 54 therapy resistance. 55 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 4

56 Chronic lymphocytic leukemia (CLL) is a biologically heterogeneous B-cell malignancy 57 characterized by progressive accumulation of CD5⁺CD19⁺ B cells in blood and lymphoid tissues. 58 Clinical behavior ranges from indolent disease to rapidly progressive forms requiring early 59 treatment[1,2]. Among established prognostic features, immunoglobulin heavy chain variable 60 region (IGHV) mutation status remains a central determinant of outcome, with IGHV-unmutated 61 CLL associated with enhanced B-cell receptor (BCR) signaling and inferior clinical 62 prognosis[3,4]. 63 CLL progression reflects both intrinsic leukemic cell programs and microenvironmental support. 64 Within lymph node and marrow niches, stromal elements, T cells, and tumor-associated 65 macrophages, including nurse-like cells (NLCs)[5,6], deliver cytokines, chemokines, and direct 66 contact signals that reinforce BCR-associated pathways, sustain metabolic fitness, and limit 67 apoptosis[7]. These niche-derived signals can attenuate responses to targeted therapies and 68 contribute to minimal residual disease[8-10]. Thus, effective therapeutic strategies may require 69 disruption of both leukemic survival pathways and macrophage-mediated niche protection. 70 Aberrant calcium signaling is a defining feature of CLL biology[11]. BCR activation promotes 71 sustained intracellular Ca²⁺ flux through store-operated calcium entry (SOCE), mediated by 72 ORAI1 and STIM channels[12]. Calcium-dependent signaling supports NFAT activation, 73 metabolic adaptation, and apoptosis resistance[13]. While proximal BCR signaling has been 74 successfully targeted with BTK inhibitors, emerging evidence suggests that downstream 75 calcium-dependent programs may persist despite inhibition of upstream nodes[14]. The molecular 76 effectors that couple SOCE to metabolic resilience in CLL remain incompletely defined. 77 78 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 5 Ca²⁺/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a serine/threonine kinase 79 activated by Ca²⁺/calmodulin that regulates AMPK and related metabolic stress-response 80 pathways[15]. CaMKK2 has been implicated in multiple malignancies, where it promotes tumor 81 cell survival, macrophage polarization, and tumor progression[16-22]. In myeloid cells, CaMKK2 82 contributes to tumor-associated macrophage differentiation and immunosuppressive 83 programming[23,24]. Recent work further links CaMKK2 to matrix-mediated mechanosensory 84 signaling and AKT activation, positioning it at the convergence of calcium signaling and 85 biomechanical adaptation[25]. However, the role of CaMKK2 in CLL, particularly in 86 coordinating leukemic survival with macrophage-mediated niche support, has not been defined. 87 We hypothesized that CaMKK2 functions as a calcium-responsive signaling hub that integrates 88 intrinsic leukemic survival programs with macrophage-mediated microenvironmental protection. 89 To test this, we examined CaMKK2 expression in clinically annotated CLL cohorts, evaluated its 90 association with disease progression, and assessed the impact of pharmacologic inhibition on 91 primary CLL cells and NLC differentiation. Our findings identify CaMKK2 as a dual-92 compartment regulator of CLL persistence and a potential therapeutic target at the interface of 93 leukemic fitness and niche support. 94 95 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 6

96 CLL patient blood samples. Peripheral blood samples were obtained from CLL patients under 97 Institutional Review Board-approved protocols Pro00011267at Duke University and the Durham 98 VA Medical Center. 99 Patients were either treatment-naïve or had not received therapy within two years prior to 100 peripheral blood collection. Available clinical and laboratory variables included age, sex, date of 101 diagnosis, Rai stage, treatment history, cytogenetic abnormalities assessed by fluorescence in situ 102 hybridization (FISH), and molecular prognostic markers, including IGHV mutation status, ZAP-103 70 expression, and CD38 expression. Clinical and laboratory characteristics of the patient 104 cohorts are summarized in Supplemental Tables 1 and 3, and in the corresponding figure 105 legends. 106 For analyses of CaMKK2 expression and clinical outcomes, a cohort of 40 patients was 107 assembled and intentionally balanced by IGHV mutation status (20 IGHV-mutated and 20 108 IGHV-unmutated cases). IGHV status represents a major biological and prognostic determinant 109 in CLL, reflecting differences in B-cell receptor signaling strength and disease 110 aggressiveness[26]. This balanced design minimized subtype-driven bias, enabled direct 111 comparison between risk groups, and permitted evaluation of CaMKK2 associations independent 112 of cohort imbalance. 113 B lymphocytes were isolated from whole blood by negative selection using the RosetteSep™ 114 Human B Cell Enrichment Cocktail (STEMCELL Technologies) and cryopreserved at -80 °C for 115 RNA isolation and quantitative RT-PCR (qRT-PCR ) analysis, as described in the corresponding 116 section. 117 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 7 Generation of nurse-like cells (NLCs). NLCs were generated from CLL Peripheral blood 118 mononuclear cells (PBMCs) as previously described[6]. Briefly, PBMC were isolated from 119 DMSO-cryopreserved CLL PBMCs by density-gradient centrifugation using Ficoll-Paque 120 (Sigma-Aldrich). PBMCs were resuspended in RPMI 1640 supplemented with 15% fetal calf 121 serum and 1% penicillin-streptomycin and glutamine. PBMCs were then plated at high density (2 122 × 10⁷ cells/mL) in tissue culture-treated plates (Corning) and maintained at 37 °C in a humidified 123 5% CO₂ atmosphere to allow differentiation of adherent NLCs. Where indicated, cultures were 124 treated at the time of plating (day 1) with the CaMKK2 inhibitor STO-609 (5 µM; Tocris) or an 125 equal volume of vehicle (Veh; DMSO). Supplemental doses of STO-609 (2.5 µM) or vehicle 126 were added on days 5 and 10. To minimize drug accumulation while preserving early adherent 127 precursors, partial medium changes were performed during treatment. After 14 days, non-128 adherent CLL-enriched cells were collected for analysis, and adherent cells were extensively 129 washed with PBS to remove residual non-adherent cells. Adherent NLCs were then imaged 130 directly by optical microscopy and subsequently detached using Macrophage Detachment 131 Solution (PromoCell), according to the manufacturer’s instructions, and used for flow-cytometric 132 phenotyping, or processed for RNA isolation and qRT-PCR, as indicated. 133 Cell lines and preparation of Tumor -Conditioned Medium (TCM). To model B -cell 134 malignancy-derived factors within the tumor microenvironment , we used tumor-conditioned 135 medium from human B cell tumor cell lines including OPM2, a multiple myeloma cell line derived 136 from a patient with plasma cell leukemia and is commonly used as a model of aggressive plasma 137 cell malignancy [27]; BJAB from Burkitt lymphoma [28], and SU-DHL-4, a diffuse large B -cell 138 lymphoma originally derived from the peritoneal effusion of a 38-year-old male with non-Hodgkin 139 lymphoma[29]. Briefly, tumor cells growing in logarithmic growth phase with viability >95% were 140 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 8 harvested, washed twice with PBS, and resuspended in fresh complete medium at a density of 1-2 141 × 10⁶ cells/mL. Cultures were incubated at 37°C in a humidified 5% CO₂ atmosphere for an 142 additional 24-48h. Cell supernatants were then collected, centrifuged to remove cells, filtered 143 through a 0.22µm membrane, aliquoted, and stored at -80 °C until use. 144 Generation of Monocyte-Derived Macrophages (MDM) from healthy donor leukocytes. 145 Leukocytes from healthy donors were obtained from the Gulf Coast Regional Blood Center as 146 de-identified commercial human blood samples. Donor leukocytes were derived from single 147 units of whole blood by density-gradient centrifugation. Donors were ≥16 years of age and met 148 standard eligibility criteria for blood donation. All identifying information was removed prior to 149 sample transfer, and samples were approved for in vitro research use; therefore, this protocol was 150 determined to be IRB-exempt. Monocyte-derived macrophages were generated using a modified 151 version of a previously described protocol[30]. Briefly, PBMC were resuspended in Monocyte 152 Attachment Medium (PromoCell) and seeded into 12-well tissue culture plates at a density of 2-4 153 × 10⁶ cells per well. Cells were incubated at 37°C in a humidified 5% CO₂ atmosphere for 1-2 h 154 to allow monocyte adherence. Non-adherent cells were removed by extensive washing and 155 adherent monocyte-enriched cells were cultured in macrophage differentiation medium (RPMI 156 1640 supplemented with recombinant human M-CSF 20 ng/mL; PeproTech), in the absence or 157 presence of tumor-conditioned media (TCM; 50% v/v). STO-609 (5 µM) or an equal volume of 158 vehicle (DMSO) was added at the time of plating (day 1). On day 3, culture media were partially 159 replaced with fresh macrophage differentiation medium containing TCM or regular medium, 160 with STO-609 or vehicle, corresponding to initial treatment conditions. After 6 days of 161 differentiation, cultures were extensively washed with PBS, and adherent MDM were detached 162 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 9 as described for NLC preparation. MDM immunophenotype was assessed by flow cytometry, 163 and aliquots of cells were processed for quantitative RT-PCR analysis. 164 MTS and Annexin V/PI assays. Primary CD19⁺ CLL cells (0.25 x 106/well) were cultured in 165 96-well/plate in 0.1 ml of Hybridoma SFM (Gibco), as described[31-33]. Cells were treated with 166 increasing concentrations of the CaMKK2 inhibitors STO-609[34] (Tocris), CC-8977[35] (Small 167 Molecule Synthesis Facility Duke University), SGC-CaMKK2-1 (Sigma)[36], or an equal volume 168 of vehicle (DMSO). Cells were then incubated at 37°C in a humidified 5% CO₂ atmosphere, and 169 CLL viability was assessed at 72 hours using two orthogonal approaches. Cytotoxicity assays 170 were performed using the MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- 171 (4-sulfophenyl)-2H-tetrazolium] assay (CellTiter 96 Aqueous One Solution Cell Proliferation 172 Assay; Promega), as described[37]. Briefly, MTS reagent was added to cell culture media and 173 incubated for four hours before cell lysis by 1% SDS. Absorbance at 490 nm was measured 174 using a microplate reader. The percentage of viable cells was determined by comparing the 175 absorbance in drug-treated cells to the absorbance in vehicle-treated cells and calculate EC50 176 values. In parallel, apoptosis/cell death was quantified by flow cytometry using Annexin V-FITC 177 (BD Biosciences) and propidium iodide (PI; Sigma) staining; total Annexin V⁺ events (PI⁻ and 178 PI⁺ populations) were quantified across the same concentration range. 179 Bright-field microscopy and ImageJ particle analysis. After 14 days of CLL-PBMC culture, 180 non-adherent cells were removed, and wells were extensively washed with PBS to eliminate 181 residual suspension cells. Adherent cells were imaged by bright-field optical microscopy using 182 an Axiovert 200 microscope (Carl Zeiss Microscopy, Thornwood, NY) and identical acquisition 183 settings across experimental conditions. For quantitative analysis, images were imported into 184 ImageJ (NIH), and adherent structures were segmented using a consistent preprocessing and 185 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 10 thresholding workflow applied uniformly across all images within an experiment. Particle 186 abundance was quantified using the ImageJ Analyze Particles function and reported as particle 187 number per unit area (particles/mm²). 188 NLC-CLL co-culture assay. NLC from CLL PBMC were generated in the presence of vehicle 189 or STO-609 (NLC-Veh and NLC-STO, respectively) as described above. After 14 days, CD19+ 190 CLL were purified from the vehicle treated groups. purified. NLC-adherent cells from Vehicle 191 and STO-609 groups were extensively washed, detached, and dispensed in 48-well plates at 1 × 192 10⁵ cells/well. CD19⁺ CLL cells (0.25 x 105/well) were then cultured either alone (“None”) or 193 co-cultured with autologous NLC-Veh or NLC-STO. At the indicated time points, non-adherent 194 cells were collected, stained with CD19, and cell viability was quantified by flow cytometry as 195 live CD19⁺ events 196 Flow cytometry. Cells were harvested at the indicated time points, washed in PBS containing 197 2% FCS, and stained with fluorochrome-conjugated antibodies for surface markers, with 198 inclusion of a fixable viability dye to exclude dead cells. All antibodies were used following 199 manufacturer’s instructions. To assess CLL yield and viability, non-adherent cells were collected 200 and stained with PE anti-human CD19 (clone HIB19, BioLegend) and live fixable dye (Zombie 201 Near-IR; BioLegend). Adherent cells (NLC or MDM) were gently detached from wells using 202 Monocyte Detachment Media (PromoCell), stained with Ig blocker, followed by saturating 203 amounts of the following antibodies for 30 minutes at 4°C: anti-human AF488 anti-human HLA-204 DR (clone L234, BioLegend), PerCP/Cy5.5 anti-human/mouse CD11b (clone M1/70; 205 BioLegend) or PE anti-human CD14 (clone HCD14, BioLegend) and live fixable dye (Zombie 206 Near-IR; or Zombie yellow, BioLegend). Cells were fixed in 4% paraformaldehyde (PFA, 207 Sigma), permeabilized with Tween-20, re-stained with PE/Cy7 anti-human CD68 (clone 208 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 11 eBioY1/82A, eBioScience) and APC anti-human CD163 (clone GHI/61 eBioScience). All 209 antibodies and staining procedures were performed according with manufacturer’s instructions. 210 Unstained and single-stained controls were used for instrument setup and compensation, and 211 fluorescence-minus-one (FMO) controls (with isotype controls as needed) were used to define 212 gating thresholds. Cells were acquired with a BD FACSCanto cytometer and analyzed using 213 FloJo 10.10.0 software. 214 Gene expression analysis. Following PBMC culture, wells were washed and adherent cells were 215 imaged as described above. Adherent cells were lysed directly in Buffer RLT (Qiagen) and 216 collected by gentle scraping. Lysates were homogenized using QIAshredder columns (Qiagen) 217 and total RNA was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s 218 instructions. cDNA was synthesized from purified RNA using the SensiFAST cDNA Synthesis 219 Kit (BIOLINE). Quantitative RT-PCR was performed using SYBR Green master mix, gene-220 specific forward and reverse primers (Integrated DNA Technologies), and cDNA template on an 221 Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System. Primers sequences are 222 listed in the Supplementary Table 4. 223 Statistical analysis. Statistical analyses were performed using GraphPad Prism (version 10.6.1). 224 Normality was assessed using Shapiro-Wilk testing and data distribution was not assumed to be 225 normal. Overall survival and time-to-treatment were analyzed by Kaplan-Meier methods and 226 compared using the log-rank (Mantel-Cox) test. For the independent validation cohort, survival 227 analysis was performed using the SurvExpress platform[38]. For primary CLL drug-sensitivity 228 assays, MTT dose-response curves were fit by nonlinear regression to derive EC50 values. 229 Associations between CaMKK2 expression and STO-609 potency, and between metabolic 230 inhibition and Annexin V-defined cell death, were assessed using Spearman rank correlation. For 231 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 12 paired analyses of matched patient samples, statistical significance was assessed using two-tailed 232 paired tests. For co-culture experiments with multiple conditions and time points, statistical 233 significance was determined by two-way ANOVA with appropriate multiple-comparisons 234 correction. For tumor-conditioned media experiments, differences across conditions were 235 evaluated by ANOVA with multiple-comparisons testing, including planned comparisons of 236 vehicle versus STO-609 within each conditioned-media condition. Unless otherwise stated, all 237 tests were two-sided. 238 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 13

239 Elevated CAMKK2 expression associates with high-risk CLL biology and clinical course. To 240 evaluate CaMKK2 as a candidate biomarker in CLL in the context of established prognostic 241 factors, we analyzed clinically annotated CLL biorepository specimens available at the Division 242 of Hematological Malignancies and Cellular Therapy (Duke University). We selected 40 243 cryopreserved peripheral blood CLL samples, balanced a priori by IGHV mutation status (20 244 IGHV-mutated; 20 IGHV-unmutated), and quantified CaMKK2 mRNA in purified CD19⁺ CLL 245 cells by qRT-PCR. Cohort characteristics are summarized in Supplementary Table 1. Because 246 CaMKK2 quantification required adequate RNA and complete clinical linkage, CaMKK2 247 expression was evaluable in 33 samples. Patients were stratified using the cohort median of 248 CaMKK2 expression (high versus low; group sizes shown in the plots), a pragmatic and unbiased 249 threshold that enables balanced comparison without data-driven optimization. Using this cutoff, 250 CaMKK2-high status was associated with significantly shorter time-to-treatment (log-rank p = 251 0.0001; Figure 1A) and inferior overall survival (log-rank p = 0.0385; Figure 1B), supporting its 252 potential clinical relevance as a biomarker of adverse outcome. CaMKK2 expression, normalized 253 by ACTB, was significantly higher in IGHV-unmutated compared with IGHV-mutated CLL 254 (Figure 1C). Further, the Duke cohort recapitulated canonical IGHV-linked biology, with higher 255 CD38 and ZAP-70 in IGHV-unmutated cases (Supplementary Figure S1). In an independent 256 dataset (GSE22762; Herold et al.[39]), CaMKK2-high expression showed a directionally 257 consistent adverse association with survival (SurvExpress[38]; Figure 1D). Finally, in a 258 multivariable model including IGHV status and Rai stage, IGHV-unmutated status remained the 259 dominant correlate of higher CaMKK2 expression (β = 0.496, 95% CI 0.310-0.682, p = 1.68 × 260 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 14 10⁻⁷), whereas Rai stage was not independently associated (p = 0.307; Supplementary Table 261 S2). Collectively, these data show that CaMKK2 expression in blood CLL cells is enriched in the 262 IGHV-unmutated subtype and associates with a more aggressive clinical course. 263 Figure 1. CaMKK2 expression in CLL associates with adverse outcome and aggressive disease biology. (A,B) Kaplan-Meier analyses of time-to-treatment (A) and overall survival (B) for CLL patients stratified by CaMKK2 expression using a median split (CaMKK2-Low, n = 16; CaMKK2-High, n = 17). Tick marks indicate censored observations. P values were calculated using the log-rank (Mantel-Cox) test. (C) CaMKK2 mRNA expression in CD19⁺ CLL cells obtained from the Duke CLL biorepository, quantified by qRT-PCR and normalized to ACTB. Samples are grouped by IGHV mutation status (IGHV-mutated [M-CLL] vs IGHV-unmutated [U-CLL]). Each symbol represents one patient; horizontal bars indicate mean ± SEM. Statistical significance was assessed by two-tailed Mann–Whitney test (**** p < 0.0001). (D) Independent cohort validation using the CLL dataset reported by Herold et al. (GSE22762). Overall survival was analyzed using the SurvExpress platform, stratifying cases into CaMKK2-high versus CaMKK2-low groups. Log-rank p value and hazard ratio (HR) with 95% confidence interval (CI) are indicated. 0 100 200 300 400 500 0 20 40 60 80 100 Time-to-Treatment Time (months) Probability CaMKK2-Low (n =16) CaMKK2-High (n =17) Logrank p= 0.0001 M-CLL U-CLL 0.00 0.05 0.10 0.15CaMKK2/ Actin ✱✱✱✱ A B C D 0 100 200 300 400 500 0 20 40 60 80 100 Survival Time (months) Probability CaMKK2-L (n =16) CaMKK2-H (n =17) Logrank p= 0.0385 0 20 40 60 80 0 20 40 60 80 100 CLL GSE22762 Time (months) Probability of Survival CaMKK2-Low (n =63) CaMKK2-High (n=44) Logrank p= 0.001743 HR = 2.66 (95% CI 1.15-6.17) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 15 Pharmacologic CaMKK2 inhibition with STO-609 reduced primary CLL viability and 264 induced Annexin V-defined cell death in a dose-dependent manner. To test whether 265 CaMKK2 represents a therapeutic vulnerability in CLL, purified primary CD19⁺ CLL cells from 266 nine independent patient samples (clinically annotated in Supplementary Table S3) were 267 treated with increasing concentrations of the CaMKK2 inhibitor STO-609 and evaluated using 268 orthogonal assays of metabolic activity (MTS) and cell death (Annexin V/PI) (Figure 2). 269 Representative dose-response curves from three samples (IDs 824-003, 400-013, and 558-013) 270 demonstrated a concentration-dependent reduction in MTS signal accompanied by an increase in 271 total Annexin V⁺ events (including PI⁻ and PI⁺ populations) (Figure 2A,B). Across the nine 272 samples, STO-609 sensitivity varied (MTS-derived EC50 range ~0.97-7.67 µM, providing 273 clinically annotated context for inter-sample heterogeneity in response. Consistent with on-target 274 biology, STO-609 potency correlated with CaMKK2 expression measured by qRT-PCR 275 (CaMKK2/ACTB), with higher CaMKK2 associated with greater sensitivity (Spearman r = -0.85, 276 p = 0.0061; Figure 2C). To distinguish STO-609-induced cytotoxicity from isolated metabolic 277 suppression, we compared MTS response and Annexin V response at a fixed dose. At 5 µM 278 STO-609, MTS viability (% of vehicle control) inversely correlated with the increase in total 279 Annexin V positivity (treated - vehicle; normalized to each sample’s maximal response at 20 280 µM) (Spearman r = -0.9333, p = 0.0007; Figure 2D). To further assess whether the observed 281 effects were specific to STO-609, two additional structurally distinct CaMKK2 inhibitors, SGC-282 CaMKK2-1[36] and CC-8977[35], were evaluated in CLL primary cells from three independent 283 patients, including one with documented multi-drug-resistant disease (Supplementary Table S4, 284 and annotated in Supplementary Table S3). Both compounds reduced CLL survival in MTS 285 assay, demonstrating activity broadly comparable to STO-609, although potency varied across 286 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 16 samples. Notably, CC-8977 exhibited higher EC50 in the multi-drug-resistant sample. These 287 findings provide supportive evidence functional evidence that inhibition of CaMKK2 activity 288 compromises the survival of primary CLL cells beyond a single inhibitor scaffold. The 289 integration of clinical associations and pharmacologic data supports CaMKK2 as both a 290 biomarker of adverse disease biology and a tractable therapeutic target in high-risk CLL. 291 Figure 2. CaMKK2 inhibition reduces viability and induces apoptosis in primary CLL cells. Primary CD19⁺ purified CLL cells (n = 9 independent patient samples) were treated with increasing concentrations of the CaMKK2 inhibitor STO-609 and assessed using complementary assays of metabolic activity and cell death. (A) Representative MTS dose-response curves from three CLL samples (IDs 824-003, 400-013, and 558-013); absorbance is expressed as percentage of vehicle control. (B) Representative flow- cytometry dose-response curves from the same samples, reporting total Annexin V⁺ cells (PI⁻ and PI⁺ populations). (C) Relationship between metabolic inhibition and cell death at 5 µM STO-609, plotting MTS (% of control) versus Δ total Annexin V⁺ (treated - vehicle), normalized to the maximal response observed at 20 µM for each sample. (D) Association between STO-609 potency and CaMKK2 expression across primary CLL samples (n = 9), plotting MTS-derived EC50 values versus CaMKK2 expression measured by qRT-PCR and normalized to ACTB. Associations were assessed using Spearman rank correlation; regression lines are shown for visualization. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 17 CaMKK2 inhibition during PBMC culture reduces CLL cell viability and alters NLC yield and 292 phenotype. CLL persistence is supported by both cell-autonomous survival programs within the 293 leukemia cell and cell-extrinsic cues provided by the myeloid microenvironment. NLCs provide 294 an established ex vivo model of this protective niche[6], while CaMKK2 is highly expressed in 295 pro-tumoral macrophage states[23,25,40], raising the possibility that CaMKK2 blockade could 296 disrupt the CLL-NLC cross-functional axis that sustains leukemic survival. To test this 297 hypothesis, PBMCs from CLL patients were cultured at high density for 14 days in the presence 298 of vehicle or STO-609 (5 µM) to generate adherent NLC (Figure 3; and annotated in 299 Supplementary Table 3). Non-adherent and adherent cells were recovered as described in 300

section and analyzed for yield, phenotype, and gene expression. 301 Compared with vehicle, STO-609 treatment significantly reduced the percentage of viable 302 CD19⁺ CLL cells recovered in the non-adherent fraction across paired patient samples (Figure 303 3A-B). Bright-field imaging after extensive washing of well plates showed reduced adherent cell 304 yield with STO-609, which was confirmed by ImageJ-based particle quantification (Figure 3B). 305 Phenotypic analysis of detached adherent cells demonstrated a decrease in the proportion of 306 CD163⁺ cells within the CD68⁺ macrophage compartment (Figure 3C), consistent with impaired 307 acquisition of an NLC-like phenotype (Figure 3C). At the transcript level, CaMKK2 was 308 detectable in CLL and, in paired samples, was expressed at higher levels in NLC than in 309 autologous CLL cells (Supplementary Figure S2). STO-609 treatment altered expression of 310 macrophage-associated inflammatory mediators (IL6 and CXCL10)[41-44], which are positively 311 associated with leukemia cell survival and disease progression, whereas CaMKK2, BAFF, and 312 APRIL mRNA levels in adherent NLC were not significantly changed (Figure 3D). Collectively, 313 these data show that CaMKK2 blockade during PBMC culture reduces CLL cell persistence 314 while limiting the formation of an adherent, CD163⁺ NLC compartment, supporting CaMKK2 as 315 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 18 a modifiable cross-cell pathway required for establishment of a pro-tumoral, protective niche in 316 CLL. 317 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 19 Figure 3. CaMKK2 inhibition during PBMC culture reduces leukemia cells viability and nurse-like cells (NLC) generation. Peripheral blood mononuclear cells (PBMCs) from CLL patients were cultured at high density for 14 days in the presence of vehicle (Veh; DMSO) or the CaMKK2 inhibitor STO-609 (5 µM). Non-adherent and adherent fractions were analyzed separately. (A) Non-adherent cells were stained for CD19 and viability markers, and CD19⁺ CLL cell viability was quantified by flow cytometry. Representative plots are displayed on the left. Paired data from individual patients are shown on the right. (B) Adherent cells were visualized by bright-field microscopy after extensive washing. (Left) Representative images are shown; scale bar = 75 µm. Insets indicate regions shown at higher magnification. Images were analyzed using ImageJ, and the mean number of adherent particles per mm² is quantified (Right). (C) Adherent cells were detached and analyzed by flow cytometry for macrophage markers. Representative histograms of CD68 and CD163 expression are shown. The right panel quantifies the percentage of CD163⁺ cells within the CD68⁺ population. (D) RNA isolated from adherent cells was analyzed by qRT-PCR to quantify expression of genes of interest normalized to ACTNB and expressed as 2⁻ΔCt. Each line represents paired measurements from an individual patient. Statistical significance was assessed using paired two-tailed tests; *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 20 STO-609 Impairs NLC-Mediated Support of CLL Cell Survival. Building on our findings 318 that CaMKK2 expression stratifies CLL clinical behavior (Figure 1; Supplemental Table S1) 319 and that pharmacologic CaMKK2 inhibition directly compromises primary CLL viability 320 (Figure 2), we next evaluated whether CaMKK2 blockade also weakens CLL survival under 321 conditions of myeloid microenvironmental support. PBMCs from CLL patients were cultured at 322 high density for 14 days with vehicle or STO-609 (5 µM) to generate adherent NLC, after which 323 NLC from vehicle- or STO-609-treated cultures were detached, replated, and used as feeder cells 324 for autologous CD19⁺ CLL cells purified from the vehicle condition (Figure 4, and annotated in 325 Supplementary Table 3). As expected, CLL cells cultured alone (“None”) showed progressive 326 loss of viability over time, whereas co-culture with vehicle-derived NLC (NLC-Veh) increased 327 recovery of live non-adherent CD19⁺ cells at days 3 and 7, confirming the protective function of 328 NLC in this system (Figure 4A,B). In contrast, NLC generated in the presence of STO-609 329 (NLC-STO) exhibited a markedly reduced capacity to sustain CLL viability, resulting in 330 significantly fewer live CD19⁺ cells compared with NLC-Veh at both time points (Figure 4A,B). 331 These effects were reproducible across three independent CLL patient samples, and two-way 332 ANOVA (time × feeder condition) with multiple-comparison correction, supporting a significant 333 impact of NLC conditioning on CLL survival (Figure 4C). Together, these data demonstrate that 334 CaMKK2 blockade impairs the pro-survival function of NLC, disrupting CLL-NLC crosstalk 335 and weakening formation of a macrophage-dependent protective niche that sustains leukemic 336 cell persistence. 337 338 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 21 Figure 4. Blocking CaMKK2 during PBMC culture impairs NLC-mediated support of CLL cell survival. PBMCs from CLL patients were cultured at high density for 14 days with vehicle or STO-609 (5 µM) to generate adherent NLC. CD19⁺ CLL cells were purified from vehicle cultures, while NLC from vehicle- or STO-609- treated cultures were detached and replated. Purified CLL cells were cultured alone (“None”) or co-cultured with NLC generated under vehicle or STO-609 conditions. (A) Representative flow-cytometry plots of viable CD19⁺ CLL cells at days 3 and 7 from a representative patient (CLL 791). (B) Quantification of live CD19⁺ CLL cells for CLL 791 at the indicated time points. (C) Summary of live CD19⁺ CLL cell percentages from three independent CLL patients. Data are shown as mean ± SEM. Statistical significance was determined by two-way ANOVA with multiple- comparison correction; **, ***, and **** indicate p < 0.01, p < 0.005, and p < 0.001, respectively. A B C NLC-VehNone NLC-STO Day 3 Day 5 Day 9 0 20 40 60 80Live CD19+ CLL (% of Total) None NLC-Veh NLC-STO ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 22 Tumor-conditioned media drives an immunosuppressive macrophage phenotype that is 339 attenuated by CaMKK2 inhibition. To test whether CaMKK2 contributes to tumor-driven 340 programming of macrophage phenotypes relevant to the CLL microenvironment, we generated 341 monocyte-derived macrophages (MDM) from 3 independent healthy donor leukocytes and 342 cultured them either in control conditions (“None”) or in the presence of tumor-conditioned 343 media (TCM) from the B-cell malignancy cell lines OPM2, SH-DHL-4, or BJAB, with vehicle 344 or STO-609 (5 µM) (Figure 5). Flow-cytometric analysis of CD68⁺ macrophages showed that 345 exposure to TCM increased the emergence of a CD163⁺ subset (Figure 5A-C), consistent with 346 induction of a pro-tumoral, NLC-like phenotype by tumor-derived signals. Across conditioned 347 media sources, CaMKK2 blockade with STO-609 attenuated this response, reducing CD163 348 expression and/or the frequency of CD163⁺ macrophages relative to matched vehicle controls 349 (Figure 5B,C), while preserving the overall CD68⁺ macrophage compartment (Figure 5A,B). 350 Together with the patient-derived NLC data (Figures 3-4), these findings support a model in 351 which CaMKK2 functions as a tractable signaling node linking tumor-derived cues to 352 macrophage polarization and microenvironmental support, thereby providing a mechanistic basis 353 for how CaMKK2 inhibition can disrupt pro-tumoral myeloid programming and weaken the 354 protective niche that sustains CLL. 355 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 23 Figure 5. Tumor-derived signals induce an immunosuppressive NLC phenotype that is attenuated by CaMKK2 inhibition. Monocyte-derived macrophages from healthy donors were cultured in the absence (“None”) or presence of tumor-conditioned media, with vehicle or STO- 609 (5 µM), and analyzed by flow cytometry. (A) Representative flow-cytometry plots showing HLA-DR and CD163 expression on CD14⁺CD68⁺ macrophages cultured with or without OPM2-conditioned medium (OPM2-CM). (B,C) Quantification of total CD68⁺ macrophages and the CD163⁺ subset following exposure to conditioned media from OPM2, SU-DHL-4, or BJAB cells. CaMKK2 inhibition limits acquisition of a CD163⁺ immunosuppressive phenotype induced by tumor-derived factors. Data represent paired analyses from independent experiments. Statistical significance was determined using paired tests; significance levels are indicated in the figure. OPM2-CM None Veh STO-609 FITC-HLA-DR APC-CD163 A B None OPM2 SU-DHL-4 BJAB Vehicle STO-609 None OPM2 SU-DHL-4 BJAB C (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 24

356 In this study, we identify CaMKK2 as a calcium-responsive signaling node that operates across 357 both leukemic and microenvironmental compartments in CLL. Our data support a model in 358 which CaMKK2 integrates intrinsic leukemic survival pathways with macrophage-mediated 359 niche support, thereby contributing to disease persistence and therapeutic resistance. 360 At the tumor cell level, elevated CaMKK2 expression in purified CD19⁺ CLL cells was 361 associated with shorter time-to-treatment and inferior overall survival and was enriched in 362 IGHV-unmutated disease. Given that IGHV-unmutated CLL exhibits heightened and sustained 363 BCR signaling and enhanced calcium flux[3,4], these findings suggest that CaMKK2 marks a 364 calcium-dependent, metabolically resilient leukemic state. 365 Mechanistically, store-operated calcium entry (SOCE) via ORAI1/STIM channels sustains 366 intracellular Ca²⁺ flux that promotes NFAT activation and apoptosis resistance[12,45]. CaMKK2 367 functions downstream of Ca²⁺/calmodulin to activate AMPK metabolic stress-adaptation 368 programs[15], providing a mechanistic link between calcium entry and mitochondrial 369 fitness[24,46]. The positive correlation between CaMKK2 expression and STO-609 sensitivity 370 reinforce the concept that CaMKK2 functions as a downstream amplifier of SOCE-mediated 371 survival signaling. Notably, recent structural analyses of aggressive stereotyped CLL subset 1 372 indicated that autonomous BCR-BCR homotypic signaling is not universally conserved, 373 suggesting that alternative mechanisms must sustain intracellular signaling and leukemic fitness. 374 In this context, CaMKK2-dependent signaling may represent a parallel or compensatory pathway 375 reinforcing disease progression. Unlike BTK inhibitors, which target proximal BCR signaling 376 nodes, CaMKK2 inhibition disrupts calcium-driven metabolic resilience, identifying a 377 mechanistically distinct vulnerability. 378 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 25 CLL progression depends on intrinsic biological features of leukemia cells as well as niche-379 mediated support[47,48]. Within lymph node and marrow niches, NLCs and other myeloid 380 populations provide cytokines and direct contact signals that reinforce BCR-associated 381 pathways, sustain metabolic adaptation, and limit apoptosis. These inputs can attenuate responses 382 to targeted agents and contribute to minimal residual disease. Our data demonstrate that 383 CaMKK2 inhibition disrupts this protective axis. STO-609 reduced the yield and of CD163⁺ 384 macrophage, altered inflammatory mediator expression, and impaired macrophage-mediated 385 survival of autologous CLL cells. These findings align with prior work showing that myeloid 386 CaMKK2 regulates tumor-associated macrophage functions and promotes tumor progression in 387 breast cancer and lymphoma models[23,24]. Importantly, recent evidence linking CaMKK2 to 388 matrix-mediated mechanosensory signaling and AKT activation further expands this 389 framework[25]. Lymph node remodeling and altered extracellular matrix composition are 390 recognized features of aggressive CLL, and CaMKK2 may function at the convergence of 391 calcium signaling, metabolic adaptation, and biomechanical cues within these niches[5,7-10]. 392 Thus, CaMKK2 appears to coordinate biochemical and structural survival inputs across both 393 leukemic and myeloid compartments. 394 The dual intrinsic-extrinsic activity of CaMKK2 carries important therapeutic implications. 395 While BTK and BCL2 inhibitors disrupt key survival pathways, microenvironmental protection 396 remains a barrier to durable disease control. Increasingly evidence indicates that macrophage-397 rich, metabolically competitive, and structural remodeled niches contribute to the comparatively 398 limited durability of CAR-T cell therapy in CLL relative to other B-cell malignancies[49-51]. 399 Immunosuppressive macrophages, cytokine gradients, and matrix-associated constraints can 400 impair T-cell fitness and persistence. By simultaneously reducing leukemic metabolic resilience 401 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 26 and attenuating macrophage-mediated support, CaMKK2 inhibition may recalibrate this hostile 402 microenvironment. Although in vivo validation is required, our findings suggest that targeting 403 CaMKK2 could enhance the efficacy of both targeted agents and immune-based therapies by 404 disrupting the bidirectional crosstalk that sustains leukemic persistence. 405 Several limitations warrant consideration. STO-609 has been widely used as selective CaMKK2 406 inhibitor in pre-clinical models[23,24,52-56], and additional compounds such as SGC-CaMKK2-1 407 and CC-8977 have demonstrated activity and selectivity across various systems[35,36,57]. 408 However, pharmacologic approaches, while translationally relevant, may have off-target effects. 409 Future studies employing genetic perturbation strategies or next-generation clinical-grade 410 inhibitors will be important to define the specific contribution of CaMKK2 to CLL biology. In 411 addition, our microenvironmental experiments rely on ex vivo PBMC-derived models which 412 capture key aspects of NLC biology but do not fully reproduce the spatial and cellular 413 complexity of lymph node niches. Finally, while the cohort was intentionally balanced by IGHV 414 status, validation in larger, independently annotated datasets with multivariable modeling will be 415 necessary to confirm the prognostic utility of CaMKK2 in CLL. 416

417 CaMKK2 emerges from this study as a clinically relevant biomarker and functional regulator of 418 chronic lymphocytic leukemia. Elevated expression identifies biologically aggressive disease and 419 associates with adverse clinical outcomes, while functional inhibition reduces leukemic cell 420 viability and weakens macrophage-mediated microenvironmental support. These findings 421 position CaMKK2 at a critical junction between tumor-intrinsic survival and niche protection. 422 By linking prognostic relevance with actionable biology, CaMKK2 represents a promising 423 candidate for biomarker-guided risk stratification and therapeutic development in high-risk CLL. 424 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 27 Institutional Review Board Statement. The study was conducted in accordance with the 425 Declaration of Helsinki and approved by the Institutional Review Board of Duke University and 426 the Durham VA Medical Center (IRB protocol number Pro00011267). 427 Informed Consent Statement. Informed consent was obtained from all subjects involved in the 428 study. 429 Data Availability Statement. The datasets generated and/or analyzed during the current study 430 are available from the corresponding author upon reasonable request. Publicly available 431 transcriptomic data used for validation were obtained from the Gene Expression Omnibus (GEO) 432 under accession number GSE22762. 433 Author Contributions. Conceptualization (SJ, LR, JBW, NC); methodology (SJ, LR, ADCV, 434 FB); formal analysis (SJ, LR); writing original draft preparation (SJ, LR, JBW, VV, DJK, NC); 435 All authors have read and agreed to the published version of the manuscript. 436 Funding. This work was supported by [Grant Agency], grant number [XXXX]. 437 Conflicts of Interest. The authors declare no conflict of interest. 438 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 24, 2026. ; https://doi.org/10.64898/2026.02.23.707451doi: bioRxiv preprint 28

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